Maximizing Product Formation and Minimizing Degradation: A Look at Buffering Conditions and Post-Reaction Degradation for the In-Line Jaffe Reaction with Capillary Electrophoresis

Creatinine levels in blood serum are typically used to assess renal function. Clinical determination of creatinine is often based on the Jaffe reaction, in which creatinine in the serum reacts with sodium picrate, resulting in a spectrophotometrically quantifiable product. Previous work from our lab has introduced an electrophoretically mediated initiation of this reaction, in which nanoliter plugs of individual reagent solutions can be added to the capillary and then mixed and reacted. Following electrophoretic separation of the product from excess reactant(s), the product can be directly determined on column. This work aims to gain a detailed understanding of the in-capillary reagent mixing dynamics, in-line reaction yield, and product degradation during electrophoresis, with an overall goal of improving assay sensitivity. One set of experiments focuses on maximizing product formation through manipulation of various conditions such as pH, voltage applied, and timing of the applied voltage, in addition to manipulations in the identity, concentration, and pH of the background electrolyte. Through this work, it was determined that dramatic changes in local voltage fields within the various reagent zones lead to ineffective reagent overlapping. Use of the software simulation program Simul 5 enabled visualization of the reaction dynamics within the capillary, specifically the wide variance between the electric field intensities within the creatinine and picrate zones. Because of this simulation work, the experimental method was modified to increase the ionic strength of the creatinine reagent zone to lower the local voltage field, thus producing more predictable and effective overlap conditions for the reagents and allowing the formation of more Jaffe product. As second set of experiments focuses on controlling the post-reaction product degradation. In that vein, we have systematically explored the importance of the identity, concentration, and pH of the background electrolyte on the post-reaction degradation rate of the product. Although prior work with borate background electrolytes indicated that product degradation was probably a function of the ionic strength of the background electrolyte, this work with a glycine background electrolyte demonstrates that degradation is in fact not a function of ionic strength of the background electrolyte. As the concentration and pH of the glycine background increased, the rate of degradation of product did not change dramatically, whereas in borate-buffered systems, the rate of Jaffe product degradation increased linearly with background electrolyte concentration above 100.0 mM borate. Similarly, increasing pH of the glycine background electrolyte did not result in a corresponding increase in product degradation, as it had with the borate background electrolyte. Other general trends that were observed include: increasing background electrolyte concentration increases peak efficiency and higher pH favors product formation; thus, it appears that use of a background electrolyte other than borate, such as glycine, the rate of degradation of the Jaffe product can be slowed, increasing the sensitivity of this in-line assay.

Radiologic identification of disaster victims: a simple and reliable method using CT of the paranasal sinuses

To assess the reliability of radiologic identification using visual comparison of ante and post mortem paranasal sinus computed tomography (CT).

Endobronchial donor pre-treatment with ventavis: is a second administration during reperfusion beneficial to optimize post-ischemic function of non-heart beating donor lungs?

BACKGROUND: Lung retrieval from non-heart-beating donors (NHBD) has been introduced into clinical practice successfully. However, because of potentially deleterious effects of warm ischemia on microvascular integrity, use of NHBD lungs is limited by short tolerable time periods before preservation. Recently, improvement of NHBD graft function was demonstrated by donor pre-treatment using aerosolized Ventavis (Schering Inc., Berlin, Germany). Currently, there is no information whether additional application of this approach in reperfusion can further optimize immediate graft function. MATERIAL AND METHODS: Asystolic pigs (n = 5/group) were ventilated for 180-min of warm ischemia (groups 1-3). In groups 2 and 3, 100 microg Ventavis were aerosolized over 30-min using an ultrasonic nebulizer (Optineb). Lungs were then retrogradely preserved with Perfadex and stored for 3-h. After left lung transplantation and contralateral lung exclusion, grafts were reperfused for 6-h. Only in group 3, another dose of 100 microg Ventavis was aerosolized during the first 30-min of reperfusion. Hemodynamics, pO2/FiO2 and dynamic compliance were monitored continuously and compared to controls. Intraalveolar edema was quantified stereologically, and extravascular-lung-water-index (EVLWI) was measured. Statistics comprised ANOVA analysis with repeated measurements. RESULTS: Dynamic compliance was significantly lower in both Ventavis groups, but additional administration did not result in further improvement. Oxygenation, pulmonary hemodynamics, EVLWI and intraalveolar edema formation were comparable between groups. CONCLUSIONS: Alveolar deposition of Ventavis in NHBD lungs before preservation significantly improves dynamic lung compliance and represents an important strategy for improvement of preservation quality and expansion of warm ischemic intervals. However, additional application of this method in early reperfusion is of no benefit.

Post-mortem tissue sampling using computed tomography guidance

PURPOSE: Currently, in forensic medicine cross-sectional imaging gains recognition and a wide use as a non-invasive examination approach. Today, computed tomography (CT) or magnetic resonance imaging that are available for patients are unable to provide tissue information on the cellular level in a non-invasive manner and also diatom detection, DNA, bacteriological, chemical toxicological and other specific tissue analyses are impossible using radiology. We hypothesised that post-mortem minimally invasive tissue sampling using needle biopsies under CT guidance might significantly enhance the potential of virtual autopsy. The purpose of this study was to test the use of a clinically approved biopsy needle for minimally invasive post-mortem sampling of tissue specimens under CT guidance. MATERIAL AND METHODS: ACN III biopsy core needles 14 gauge x 160 mm with automatic pistol device were used on three bodies dedicated to research from the local anatomical institute. Tissue probes from the brain, heart, lung, liver, spleen, kidney and muscle tissue were obtained under CT fluoroscopy. RESULTS: CT fluoroscopy enabled accurate placement of the needle within the organs and tissues. The needles allowed for sampling of tissue probes with a mean width of 1.7 mm (range 1.2-2 mm) and the maximal length of 20 mm at all locations. The obtained tissue specimens were of sufficient size and adequate quality for histological analysis. CONCLUSION: Our results indicate that, similar to the clinical experience but in many more organs, the tissue specimens obtained using the clinically approved biopsy needle are of a sufficient size and adequate quality for a histological examination. We suggest that post-mortem biopsy using the ACN III needle under CT guidance may become a reliable method for targeted sampling of tissue probes of the body.

Increased sensitivity in mapping task demand in visuospatial processing using reaction-time-dependent hemodynamic response predictors in rapid event-related fMRI

Searching for the neural correlates of visuospatial processing using functional magnetic resonance imaging (fMRI) is usually done in an event-related framework of cognitive subtraction, applying a paradigm comprising visuospatial cognitive components and a corresponding control task. Besides methodological caveats of the cognitive subtraction approach, the standard general linear model with fixed hemodynamic response predictors bears the risk of being underspecified. It does not take into account the variability of the blood oxygen level-dependent signal response due to variable task demand and performance on the level of each single trial. This underspecification may result in reduced sensitivity regarding the identification of task-related brain regions. In a rapid event-related fMRI study, we used an extended general linear model including single-trial reaction-time-dependent hemodynamic response predictors for the analysis of an angle discrimination task. In addition to the already known regions in superior and inferior parietal lobule, mapping the reaction-time-dependent hemodynamic response predictor revealed a more specific network including task demand-dependent regions not being detectable using the cognitive subtraction method, such as bilateral caudate nucleus and insula, right inferior frontal gyrus and left precentral gyrus.

Affective and linguistic processing of speech prosody: DC potential studies

Speech melody or prosody subserves linguistic, emotional, and pragmatic functions in speech communication. Prosodic perception is based on the decoding of acoustic cues with a predominant function of frequency-related information perceived as speaker's pitch. Evaluation of prosodic meaning is a cognitive function implemented in cortical and subcortical networks that generate continuously updated affective or linguistic speaker impressions. Various brain-imaging methods allow delineation of neural structures involved in prosody processing. In contrast to functional magnetic resonance imaging techniques, DC (direct current, slow) components of the EEG directly measure cortical activation without temporal delay. Activation patterns obtained with this method are highly task specific and intraindividually reproducible. Studies presented here investigated the topography of prosodic stimulus processing in dependence on acoustic stimulus structure and linguistic or affective task demands, respectively. Data obtained from measuring DC potentials demonstrated that the right hemisphere has a predominant role in processing emotions from the tone of voice, irrespective of emotional valence. However, right hemisphere involvement is modulated by diverse speech and language-related conditions that are associated with a left hemisphere participation in prosody processing. The degree of left hemisphere involvement depends on several factors such as (i) articulatory demands on the perceiver of prosody (possibly, also the poser), (ii) a relative left hemisphere specialization in processing temporal cues mediating prosodic meaning, and (iii) the propensity of prosody to act on the segment level in order to modulate word or sentence meaning. The specific role of top-down effects in terms of either linguistically or affectively oriented attention on lateralization of stimulus processing is not clear and requires further investigations.

Statistical evaluation of time-dependent metabolite concentrations: estimation of post-mortem intervals based on in situ 1H-MRS of the brain

Knowledge of the time interval from death (post-mortem interval, PMI) has an enormous legal, criminological and psychological impact. Aiming to find an objective method for the determination of PMIs in forensic medicine, 1H-MR spectroscopy (1H-MRS) was used in a sheep head model to follow changes in brain metabolite concentrations after death. Following the characterization of newly observed metabolites (Ith et al., Magn. Reson. Med. 2002; 5: 915-920), the full set of acquired spectra was analyzed statistically to provide a quantitative estimation of PMIs with their respective confidence limits. In a first step, analytical mathematical functions are proposed to describe the time courses of 10 metabolites in the decomposing brain up to 3 weeks post-mortem. Subsequently, the inverted functions are used to predict PMIs based on the measured metabolite concentrations. Individual PMIs calculated from five different metabolites are then pooled, being weighted by their inverse variances. The predicted PMIs from all individual examinations in the sheep model are compared with known true times. In addition, four human cases with forensically estimated PMIs are compared with predictions based on single in situ MRS measurements. Interpretation of the individual sheep examinations gave a good correlation up to 250 h post-mortem, demonstrating that the predicted PMIs are consistent with the data used to generate the model. Comparison of the estimated PMIs with the forensically determined PMIs in the four human cases shows an adequate correlation. Current PMI estimations based on forensic methods typically suffer from uncertainties in the order of days to weeks without mathematically defined confidence information. In turn, a single 1H-MRS measurement of brain tissue in situ results in PMIs with defined and favorable confidence intervals in the range of hours, thus offering a quantitative and objective method for the determination of PMIs.

Effects of butorphanol on the withdrawal reflex using threshold, suprathreshold and repeated subthreshold electrical stimuli in conscious horses

OBJECTIVE: To assess the effects of a single intravenous dose of butorphanol (0.1 mg kg(-1)) on the nociceptive withdrawal reflex (NWR) using threshold, suprathreshold and repeated subthreshold electrical stimuli in conscious horses. STUDY DESIGN: 'Unblinded', prospective experimental study. ANIMALS: Ten adult horses, five geldings and five mares, mean body mass 517 kg (range 487-569 kg). METHODS: The NWR was elicited using single transcutaneous electrical stimulation of the palmar digital nerve. Repeated stimulations were applied to evoke temporal summation. Surface electromyography was performed to record and quantify the responses of the common digital extensor muscle to stimulation and behavioural reactions were scored. Before butorphanol administration and at fixed time points up to 2 hours after injection, baseline threshold intensities for NWR and temporal summation were defined and single suprathreshold stimulations applied. Friedman repeated-measures analysis of variance on ranks and Wilcoxon signed-rank test were used with the Student-Newman-Keul's method applied post-hoc. The level of significance (alpha) was set at 0.05. RESULTS: Butorphanol did not modify either the thresholds for NWR and temporal summation or the reaction scores, but the difference between suprathreshold and threshold reflex amplitudes was reduced when single stimulation was applied. Upon repeated stimulation after butorphanol administration, a significant decrease in the relative amplitude was calculated for both the 30-80 and the 80-200 millisecond intervals after each stimulus, and for the whole post-stimulation interval in the right thoracic limb. In the left thoracic limb a decrease in the relative amplitude was found only in the 30-80 millisecond epoch. CONCLUSION: Butorphanol at 0.1 mg kg(-1) has no direct action on spinal Adelta nociceptive activity but may have some supraspinal effects that reduce the gain of the nociceptive system. CLINICAL RELEVANCE: Butorphanol has minimal effect on sharp immediate Adelta-mediated pain but may alter spinal processing and decrease the delayed sensations of pain.

New method for quantification and statistical analysis of nociceptive reflex receptive fields in humans

A method for quantifying nociceptive withdrawal reflex receptive fields in human volunteers and patients is described. The reflex receptive field (RRF) for a specific muscle denotes the cutaneous area from which a muscle contraction can be evoked by a nociceptive stimulus. The method is based on random stimulations presented in a blinded sequence to 10 stimulation sites. The sensitivity map is derived by interpolating the reflex responses evoked from the 10 sites. A set of features describing the size and location of the RRF is presented based on statistical analysis of the sensitivity map within every subject. The features include RRF area, volume, peak location and center of gravity. The method was applied to 30 healthy volunteers. Electrical stimuli were applied to the sole of the foot evoking reflexes in the ankle flexor tibialis anterior. The RRF area covered a fraction of 0.57+/-0.06 (S.E.M.) of the foot and was located on the medial, distal part of the sole of the foot. An intramuscular injection into flexor digitorum brevis of capsaicin was performed in one spinal cord injured subject to attempt modulation of the reflex receptive field. The RRF area, RRF volume and location of the peak reflex response appear to be the most sensitive measures for detecting modulation of spinal nociceptive processing. This new method has important potential applications for exploring aspects of central plasticity in volunteers and patients. It may be utilized as a new diagnostic tool for central hypersensitivity and quantification of therapeutic interventions.

A comparison of ground cover and frequency estimation methods for post-harvest soil monitoring

The amount and type of ground cover is an important characteristic to measure when collecting soil disturbance monitoring data after a timber harvest. Estimates of ground cover and bare soil can be used for tracking changes in invasive species, plant growth and regeneration, woody debris loadings, and the risk of surface water runoff and soil erosion. A new method of assessing ground cover and soil disturbance was recently published by the U.S. Forest Service, the Forest Soil Disturbance Monitoring Protocol (FSDMP). This protocol uses the frequency of cover types in small circular (15cm) plots to compare ground surface in pre- and post-harvest condition. While both frequency and percent cover are common methods of describing vegetation, frequency has rarely been used to measure ground surface cover. In this study, three methods for assessing ground cover percent (step-point, 15cm dia. circular and 1x5m visual plot estimates) were compared to the FSDMP frequency method. Results show that the FSDMP method provides significantly higher estimates of ground surface condition for most soil cover types, except coarse wood. The three cover methods had similar estimates for most cover values. The FSDMP method also produced the highest value when bare soil estimates were used to model erosion risk. In a person-hour analysis, estimating ground cover percent in 15cm dia. plots required the least sampling time, and provided standard errors similar to the other cover estimates even at low sampling intensities (n=18). If ground cover estimates are desired in soil monitoring, then a small plot size (15cm dia. circle), or a step-point method can provide a more accurate estimate in less time than the current FSDMP method.

Slope stability analysis of the Pacaya Volcano, Guatemala, using limit equilibrium and finite element method

The Pacaya volcanic complex is part of the Central American volcanic arc, which is associated with the subduction of the Cocos tectonic plate under the Caribbean plate. Located 30 km south of Guatemala City, Pacaya is situated on the southern rim of the Amatitlan Caldera. It is the largest post-caldera volcano, and has been one of Central America’s most active volcanoes over the last 500 years. Between 400 and 2000 years B.P, the Pacaya volcano had experienced a huge collapse, which resulted in the formation of horseshoe-shaped scarp that is still visible. In the recent years, several smaller collapses have been associated with the activity of the volcano (in 1961 and 2010) affecting its northwestern flanks, which are likely to be induced by the local and regional stress changes. The similar orientation of dry and volcanic fissures and the distribution of new vents would likely explain the reactivation of the pre-existing stress configuration responsible for the old-collapse. This paper presents the first stability analysis of the Pacaya volcanic flank. The inputs for the geological and geotechnical models were defined based on the stratigraphical, lithological, structural data, and material properties obtained from field survey and lab tests. According to the mechanical characteristics, three lithotechnical units were defined: Lava, Lava-Breccia and Breccia-Lava. The Hoek and Brown’s failure criterion was applied for each lithotechnical unit and the rock mass friction angle, apparent cohesion, and strength and deformation characteristics were computed in a specified stress range. Further, the stability of the volcano was evaluated by two-dimensional analysis performed by Limit Equilibrium (LEM, ROCSCIENCE) and Finite Element Method (FEM, PHASE 2 7.0). The stability analysis mainly focused on the modern Pacaya volcano built inside the collapse amphitheatre of “Old Pacaya”. The volcanic instability was assessed based on the variability of safety factor using deterministic, sensitivity, and probabilistic analysis considering the gravitational instability and the effects of external forces such as magma pressure and seismicity as potential triggering mechanisms of lateral collapse. The preliminary results from the analysis provide two insights: first, the least stable sector is on the south-western flank of the volcano; second, the lowest safety factor value suggests that the edifice is stable under gravity alone, and the external triggering mechanism can represent a likely destabilizing factor.

Incidence of auditory ossicle luxation and petrous bone fractures detected in post-mortem multislice computed tomography (MSCT)

As the auditory ossicles are difficult to display without harming them in conventional autopsies, lesions of these minute bones and the ossicular chain are regularly missed. In this study, the method of choice in clinical medicine for the examination of such lesions, namely multislice computed tomography, was applied to 100 corpses. The hereby obtained results regarding ossicle luxation and petrous bone fracture indicated that the lesions were not dependant on the amount, but rather on the type of energy inflicted to the head.

Gunshot injuries detected by post-mortem multislice computed tomography (MSCT): a feasibility study

Modern cross-sectional imaging techniques are being increasingly implemented in forensic pathology. These methods may serve as an adjuvant to classic forensic autopsies or even replace them altogether in the future. In order to assess the practicability of such a method, namely post-mortem multislice computed tomography (MSCT) in fatal gunshot injuries, 22 corpses underwent such an examination prior to forensic autopsy. The cardinal questions of the location of entrance and exit wounds, the detection of bullets and bullet fragments in the body, the bullet course, inflicted injuries and cause of death were addressed at MSCT and autopsy. The results of the two techniques revealed that post-mortem MSCT can answer these questions reliably and is therefore a useful tool in the assessment of such injuries.

Injuries due to sharp trauma detected by post-mortem multislice computed tomography (MSCT): a feasibility study

Modern cross-sectional imaging techniques are being increasingly implemented in forensic pathology. In order to assess the practicability of such a method, namely post-mortem multislice computed tomography (MSCT) in cases of fatal cut and stab injuries, 12 corpses underwent such an examination prior to forensic autopsy. The questions regarding detection of foreign bodies, wound channels, skeletal and organ injuries, as well as the cause of death were addressed at MSCT and autopsy. The results of the two techniques revealed that post-mortem MSCT a useful tool in the assessment of such injuries.

Non-invasive method to predict viscosity and size using total internal reflection fluorescence microscopy (TIRFM) system

This study develops an automated analysis tool by combining total internal reflection fluorescence microscopy (TIRFM), an evanescent wave microscopic imaging technique to capture time-sequential images and the corresponding image processing Matlab code to identify movements of single individual particles. The developed code will enable us to examine two dimensional hindered tangential Brownian motion of nanoparticles with a sub-pixel resolution (nanoscale). The measured mean square displacements of nanoparticles are compared with theoretical predictions to estimate particle diameters and fluid viscosity using a nonlinear regression technique. These estimated values will be confirmed by the diameters and viscosities given by manufacturers to validate this analysis tool. Nano-particles used in these experiments are yellow-green polystyrene fluorescent nanospheres (200 nm, 500 nm and 1000 nm in diameter (nominal); 505 nm excitation and 515 nm emission wavelengths). Solutions used in this experiment are de-ionized (DI) water, 10% d-glucose and 10% glycerol. Mean square displacements obtained near the surface shows significant deviation from theoretical predictions which are attributed to DLVO forces in the region but it conforms to theoretical predictions after ~125 nm onwards. The proposed automation analysis tool will be powerfully employed in the bio-application fields needed for examination of single protein (DNA and/or vesicle) tracking, drug delivery, and cyto-toxicity unlike the traditional measurement techniques that require fixing the cells. Furthermore, this tool can be also usefully applied for the microfluidic areas of non-invasive thermometry, particle tracking velocimetry (PTV), and non-invasive viscometry.