Validation of a Spectrophotometric Method for Quantification of Carboxyhemoglobin

Universidade de São Paulo - LIM[40/HC-FM-USP]

Scanning electron microscopy as an auxiliary method in the study of exhumed bones

Scanning electronmicroscopy (SEM) has been used in forensic science in many ways. The reports of cases in which SEM has been used as an auxiliary method in the investigation of exhumed bones are rare. In this article, we report an exhumation that was made to determine if a seized weapon could have been used in a homicide. We used SEM to analyze a fracture in the interior of the skull of the victim. The findings described in this article showed us that it is possible to develop new researches in this field. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

A method to immunolabel rodent spinal cord neurons and glia for molecular study in specific laser microdissected cells involved in neurodegenerative disorders

Neuron-glia interaction is involved in physiological function of neurons, however, recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Laser microdissection offers a unique opportunity to study molecular regulation in specific immunolabeled cell types. However, an adequate protocol to allow morphological and molecular analysis of rodent spinal cord astrocyte, microglia and motoneurons remains a big challenge. In this paper we present a quick method to immunolabel those cells in flash frozen sections to be used in molecular biology analyses after laser microdissection and pressure catapulting.

Collagen V and vascular injury promote lung architectural changes in systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disorder characterized by inflammation, fibrosis and vascular damage. The aim of this study was to evaluate the interactions between basement membrane disruption, endothelial injury and collagen V deposition on the vascular wall, as well as their association with pulmonary function tests in patients with SSc. Method: The endothelial apoptosis was assessed by TUNEL and electron microscopy, and quantified through the point-counting technique. To evaluate basement membrane integrity, laminin immunostaining and electron microscopy were used. Immunofluorescence and morphometric analysis were used to determine the amount of collagen V in the vascular walls in 23 open lung biopsies of patients with SSc without pulmonary hypertension. Normal lung tissue was obtained from five individuals who had died of traumatic injuries. Results: The apoptosis index in SSc was higher in the endothelial cells (13.83 +/- 6.83) when compared with the control (2.51 +/- 2.06) group (P < 0.001) and confirmed by electron microscopy. We observed an important disruption of the basement membrane on the vascular wall shown by discontinuous laminin immunostaining and electron microscopy. An increase in collagen V on the vascular wall of the SSc group was observed (45.28 +/- 13.21), when compared with control group (22.90 +/- 4.13, P < 0.001), and this difference was statistically significant. An inverse correlation was found between vital capacity, forced vital capacity, forced expiratory volume in 1 s, vascular collagen V and endothelial apoptosis (P < 0.05). Conclusions: We conclude that the endothelial apoptosis and vascular collagen V interaction reinforce the vascular pathway in the SSc pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential. Please cite this paper as: Parra ER, Aguiar AC Jr, Teodoro WR, de Souza R, Yoshinari NH and Capelozzi VL. Collagen V and vascular injury promote lung architectural changes in systemic sclerosis. The Clinical Respiratory Journal 2009; 3: 135-142.

A method for better positioning bipolar electrodes for lower limb EMG recordings during dynamic contractions

To obtain a high quality EMG acquisition, the signal must be recorded as far away as possible from muscle innervations and tendon zones, which are known to shift during dynamic contractions. This study describes a methodology, using commercial bipolar electrodes, to identify better electrode positions for superficial EMG of lower limb muscles during dynamic contractions. Eight female volunteers participated in this study. Myoelectric signals of the vastus lateralis, gastrocnemius medialis, peroneus longus and tibialis anterior muscles were acquired during maximum isometric contractions using bipolar electrodes. The electrode positions of each muscle were selected assessing SENIAM and then, other positions were located along the length of muscle up and down the SENIAM site. The raw signal (density), the linear envelopes, the RMS value, the motor point site, the position of the IZ and its shift during dynamic contractions were taken into account to select and compare electrode positions. For vastus lateralis and peroneus longus, the best sites were 66% and 25% of muscle length, respectively (similar to SENIAM location). The position of the tibialis anterior electrodes presented the best signal at 47.5% of its length (different from SENIAM location). The position of the gastrocnemius medialis electrodes was at 38% of its length and SENIAM does not specify a precise location for signal acquisition. The proposed method should be considered as another methodological step in every EMG study to guarantee the quality of the signal and subsequent human movement interpretations. (C) 2009 Elsevier B.V. All rights reserved.

Contrast-enhanced magnetic resonance imaging identifies focal regions of intramyocardial fibrosis, in patients with severe aortic valve disease: Correlation with quantitative histopathology

Background Chronic aortic valve disease (AVD) is characterized by progressive accumulation of interstitial myocardial fibrosis (MF). However, assessment of MF accumulation has only been possible through histologic analyses of endomyocardial biopsies. We sought to evaluate contrast-enhanced magnetic resonance imaging (ce-MRI) as a noninvasive method to identify the presence of increased MF in patients with severe AVD. Methods Seventy patients scheduled to undergo aortic valve replacement surgery were examined by cine and ce-MRI in a 1.5-T scanner. Cine images were used for the assessment of left ventricular (LV) volumes, mass, and function. Delayed-enhancement images were used to characterize the regions of MF. In addition, histologic analyses of myocardial samples obtained during aortic valve replacement surgery were used for direct quantification of interstitial MF. Ten additional subjects who died of noncardiac causes served as controls for the quantitative histologic analyses. Results Interstitial MF determined by histopathologic analysis was higher in patients with AVID than in controls (2.7% +/- 2.0% vs 0.6% +/- 0.2%, P =.001). When compared with histopathologic results, ce-MRI demonstrated a sensitivity of 74%, a specificity of 81%, and an accuracy of 76% to identify AVD patients with increased interstitial MF There was a significant inverse correlation between interstitial MF and LV ejection fraction (r = -0.67, P <.0001). Accordingly, patients with identifiable focal regions of MF by ce-MRI exhibited worse LV systolic function than those without MF (45% +/- 14% vs 65% +/- 14%, P <.0001). Conclusions Contrast-enhanced MRI allows for the noninvasive detection of focal regions of MF in patients with severe AVD. Moreover, patients with identifiable MF by ce-MRI exhibited worse LV functional parameters. (Am Heart J 2009; 157:361-8.)

Inhibin A Production after Gonadotropin Stimulus: A New Method to Detect Ovarian Tissue in Ovotesticular Disorder of Sex Development

Background/Aims: While laboratory methods for the detection of testicular tissue are well standardized, currently there is no available test to demonstrate the presence of ovarian tissue. We evaluated the effectiveness of gonadal stimulation with luteinizing hormone (LH)/follicle-stimulating hormone (FSH) for the detection of ovarian tissue in patients with disorders of sex development (DSD). Methods: Ten patients with congenital adrenal hyperplasia (CAH) as ovarian-positive controls, 10 with cryptorchidism (ovarian-negative controls), 13 patients with DSD of no defined etiology and 7 patients with ovotesticular DSD (true hermaphroditism, TH) were included in the study. They underwent a daily injection of both LH and FSH on 3 consecutive days. LH, FSH, estradiol, testosterone and inhibin A were measured before treatment, 24 h after the 1st dose and 24 h after the 3rd dose. Results: Estradiol increased in all CAH and TH patients, with a median value of 155.1 and 92.6 pg/ml, respectively, after the 3rd injection. Inhibin A also increased in all CAH and TH patients, with a median value of 70.4 and 32.2 pg/ml, respectively, after the 3rd injection. There was no change in these hormones in the other groups. Conclusion: The LH/FSH stimulation test might be a useful method to detect the presence of ovarian tissue. Copyright (C) 2009 S. Karger AG, Basel

Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil

Some Latin American countries have plans for total control and/or eradication of Chagas disease by the main vector (Triatoma infestans) and by blood transfusion. To achieve this, patients with Chagas disease must be identified. A Western blotting test, TESAcruzi, is described as a supplemental test for diagnosis of Chagas disease using samples collected from children <5 years living in different states of Brazil. Blood samples collected by finger prick on filter paper were sent to the test laboratory by a central laboratory to confirm results obtained previously. Ten percent of negative samples, all doubtful and all positive samples were received. Commercial reagents, IgG indirect immunofluorescence, enzyme immunoassay, and a recently introduced TESAcruzi test were used. From 8788 samples, 163 (1.85%) were reactive by IgG-ELISA and 312 (3.55%) by IgG IIF. From these, 77 (0.87%) were reactive in the TESAcruzi test. The results had high clinical value to identify those truly infected. (C) 2010 Elsevier B.V. All rights reserved.

Validity and reliability of a computer method to estimate vertebral axial rotation from digital radiographs

Axial vertebral rotation, an important parameter in the assessment of scoliosis may be identified on X-ray images. In line with the advances in the field of digital radiography, hospitals have been increasingly using this technique. The objective of the present study was to evaluate the reliability of computer-processed rotation measurements obtained from digital radiographs. A software program was therefore developed, which is able to digitally reproduce the methods of Perdriolle and Raimondi and to calculate semi-automatically the rotation degree of vertebra on digital radiographs. Three independent observers estimated vertebral rotation employing both the digital and the traditional manual methods. Compared to the traditional method, the digital assessment showed a 43% smaller error and a stronger correlation. In conclusion, the digital method seems to be reliable and enhance the accuracy and precision of vertebral rotation measurements.

An easy method for handling the genus Phoneutria (Araneae, Ctenidae) for venom extraction

This paper describes an easy, cheap, and safe method of capturing and handling the medically important spider Phoneutria for venom extraction. The method does not injure or kill the spider and allows the extraction of pure venom.

The ""single-section"" Golgi method adapted for formalin-fixed human brain and light microscopy

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The ""single-section"" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, ""sandwiched"" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the ""single-section"" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. (C) 2010 Elsevier B.V. All rights reserved.

PREDICTION OF FAT-FREE MASS BY BIOELECTRICAL IMPEDANCE ANALYSIS IN OLDER ADULTS FROM DEVELOPING COUNTRIES: A CROSS-VALIDATION STUDY USING THE DEUTERIUM DILUTION METHOD

Objective: Several limitations of published bioelectrical impedance analysis (BIA) equations have been reported. The aims were to develop in a multiethnic, elderly population a new prediction equation and cross-validate it along with some published BIA equations for estimating fat-free mass using deuterium oxide dilution as the reference method. Design and setting: Cross-sectional study of elderly from five developing countries. Methods: Total body water (TBW) measured by deuterium dilution was used to determine fat-free mass (FFM) in 383 subjects. Anthropometric and BIA variables were also measured. Only 377 subjects were included for the analysis, randomly divided into development and cross-validation groups after stratified by gender. Stepwise model selection was used to generate the model and Bland Altman analysis was used to test agreement. Results: FFM = 2.95 - 3.89 (Gender) + 0.514 (Ht(2)/Z) + 0.090 (Waist) + 0.156 (Body weight). The model fit parameters were an R(2), total F-Ratio, and the SEE of 0.88, 314.3, and 3.3, respectively. None of the published BIA equations met the criteria for agreement. The new BIA equation underestimated FFM by just 0.3 kg in the cross-validation sample. The mean of the difference between FFM by TBW and the new BIA equation were not significantly different; 95% of the differences were between the limits of agreement of -6.3 to 6.9 kg of FFM. There was no significant association between the mean of the differences and their averages (r = 0.008 and p = 0.2). Conclusions: This new BIA equation offers a valid option compared with some of the current published BIA equations to estimate FFM in elderly subjects from five developing countries.

An association rule-based method to support medical image diagnosis with efficiency

In this paper, we propose a method based on association rule-mining to enhance the diagnosis of medical images (mammograms). It combines low-level features automatically extracted from images and high-level knowledge from specialists to search for patterns. Our method analyzes medical images and automatically generates suggestions of diagnoses employing mining of association rules. The suggestions of diagnosis are used to accelerate the image analysis performed by specialists as well as to provide them an alternative to work on. The proposed method uses two new algorithms, PreSAGe and HiCARe. The PreSAGe algorithm combines, in a single step, feature selection and discretization, and reduces the mining complexity. Experiments performed on PreSAGe show that this algorithm is highly suitable to perform feature selection and discretization in medical images. HiCARe is a new associative classifier. The HiCARe algorithm has an important property that makes it unique: it assigns multiple keywords per image to suggest a diagnosis with high values of accuracy. Our method was applied to real datasets, and the results show high sensitivity (up to 95%) and accuracy (up to 92%), allowing us to claim that the use of association rules is a powerful means to assist in the diagnosing task.

Validation of a manual headspace gas chromatography method for determining volatile compounds in biological fluids

Background: We report the validation of a method for the determination of acetaldehyde, acetone, methanol, and ethanol in biological fluids using manual headspace sample introduction and an acetonitrile internal standard. Method: This method uses a capillary column (I = 30 m, I.D. = 0.25 mm, dF = 0.25 mu m) installed in a gas chromatography-flame ionization detector (GC-FID) apparatus with a run time of 7.5 minutes. Results: Analysis of the retention times and the resolution of the analyte peaks demonstrated excellent separation without widening of the peaks. Precision and accuracy were good (interassay precision < 15% and recovery between 85% and 115%) in both blood and urine. Conclusion: The method was linear (r > 0.09) over the analytical measurement range (AMR) of each analyte.