Different Methods For Quantification Research Articles

Lack of Interchangeability Between 3 Different Methods for Quantification of Everolimus in Blood: ACMIA, LTIA, and UHPLC-MS/MS.

Affinity chrome-mediated immunoassays (ACMIA) do not require pretreatment and have a wide calibration range and good analytical performance. To date, no studies have compared ACMIA and latex agglutination turbidimetry immunoassays (LTIA). The objective of this study was to evaluate the interchangeability of ACMIA, LTIA, and the previously developed ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). A total of 111 whole blood samples were collected from 25 patients undergoing routine everolimus therapeutic drug monitoring. The interchangeability between the 3 methods was assessed using robust Passing-Bablok regression analysis and Bland-Altman plots. All samples were quantifiable by UHPLC-MS/MS, whereas 56 and 1 samples were below the lower limits of quantification by LTIA and ACMIA, respectively. In the robust Passing-Bablok regression plots, the slopes of the regression equations between ACMIA and UHPLC-MS/MS, LTIA and UHPLC-MS/MS, and ACMIA and LTIA were 1.23 (95% [confidence interval] CI, 1.13-1.33), 0.67 (95% CI, 0.57-0.77), and 1.71 (95% CI, 1.43-2.33), respectively, with significant proportional biases indicating no interchangeability among all 3 methods. Bland-Altman plots also revealed statistically significant proportional biases between ACMIA and UHPLC-MS/MS (P = 0.012), LTIA and UHPLC-MS/MS (P < 0.001), and ACMIA and LTIA (P < 0.001). Statistically significant proportional biases were observed among the 3 methods. Blood everolimus concentration measurements should be interpreted with caution when switching the quantification methods for therapeutic drug monitoring.

Quantitative Late Gadolinium Enhancement Cardiac Magnetic Resonance and Sudden Death in Hypertrophic Cardiomyopathy: A Meta-Analysis

BackgroundQuantitative late gadolinium enhancement (LGE) cardiac magnetic resonance provides important prognostic information for sudden cardiac death (SCD) in hypertrophic cardiomyopathy (HCM). However, it has not been fully integrated into clinical practice. ObjectivesThe purpose of this study was to assess the prognostic value of LGE extent in predicting SCD in adults with HCM across different methods of quantification, thresholds, and patients’ clinical profile. MethodsThe authors searched PubMed, Web of Science, and Cochrane Library for studies investigating the prognostic value of LGE% in predicting SCD in HCM. Pooled ORs were calculated with 95% CIs. The optimal threshold was determined using a multiple cutoffs model. ResultsEleven studies were included in the meta-analysis with a total of 5,550 patients and a median follow-up time of 5.2 years. Two studies quantified LGE manually, 7 studies used the 6 SD technique, 1 study used the 4 SD technique, and 1 study the 2 SD technique. There was no statistically significant difference in predicting SCD between these 4 methods (P = 0.443). Optimal cutoff could be determined only for the 6 SD technique. LGE 10% was the optimal threshold of the 6 SD technique with sensitivity 0.73 and specificity 0.67. ConclusionsThe different LGE quantification techniques have comparable accuracy in predicting SCD. When the more extensively studied 6 SD technique is used, LGE 10% is the optimal cutoff and can effectively restratify intermediate-risk patients. LGE extent can improve HCM risk stratification, but it is unlikely to become a standalone tool.

Treatment with Gaseous Ozone Significantly Reduced the Number of Bacteria in Extended-Spectrum-β-Lactamase (ESBL)-Producing Escherichia coli Biofilm

Ozone is a triatomic allotropic modification of oxygen with very high oxidation potential and strong antimicrobial properties, and can be used as a disinfecting agent. The aim of this work was to investigate the effectiveness of gaseous ozone in reducing the number of bacteria and the total biomass of E. coli biofilm using different methods of quantification and detection. Biofilm of all tested clinical isolates and standard strain was grown on ceramic tiles with dimensions of 1.0 × 1.0 cm over 24 h. These plates were then treated with gaseous ozone for 1 h. After washing, CFU/cm2 was determined, ATP bioluminescence was measured with a luminometer, and the total biomass reduction was measured after crystal-violet staining. Gaseous ozone proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Treatment caused a reduction in total bacteria number of up to 2.00 log10CFU/cm2, followed by a reduction in total biomass of up to 61.40%. Inhibition rates varied from 35.80% to 99.41%, depending on the method of quantification used. All methods used in this study are effective in determining the anti-biofilm activity of gaseous ozone, but more research is needed.

Differences in Mean Values and Variance in Quantitative Analyses of Foveal OCTA Imaging.

Multiple approaches for quantifying parameters such as vessel density (VD) and vessel length density (VLD) in optical coherence tomography angiography (OCTA) en-face segmentations are currently available. While it is common knowledge that data gathered from different methods should not be directly compared to each other, a comparison of the different methods can help to further the understanding of differences between different methods of measurement. Here we compare a common method of semiautomatically quantifying VD and VLD with an automated method supplied by the manufacturer of an OCTA device and report on differences in performance in order to probe for and highlight differences in values gathered by both methods. OCTA was performed using the swept source PLEX Elite 9000 device, software version 2.0.1.47652 (Carl Zeiss Meditec Inc., Dublin, CA, USA). Scans of 3 mm × 3 mm from healthy volunteers centred on the fovea were acquired by a well-trained certified ophthalmologist. Scans with a signal strength of 8 out of 10 or higher were included. Quantitative parameters of the 3 mm × 3 mm cube scans were automatically generated and segmented into superficial capillary plexus (SCP) and deep capillary plexus (DCP) layers using layer segmentation produced by the instrument software and prototype analysis VD quantification software (Macular Density v.0.7.1, ARI Network Hub, Carl Zeiss Meditec Inc., Dublin, CA, USA) supplied by the manufacturer. An alternative approach of quantitative analysis of VD and VLD was performed manually with ImageJ (National Institutes of Health, Bethesda, Maryland, USA), as previously reported. VD was assessed as the ratio of the retinal area occupied by vessels. VDL was measured as the total length of the skeletonised vessels using 1-pixel centre line extraction of the blood vessels. We report differences in standard deviation (SD) in OCTA parameters obtained using different methods. The standard deviation of VD and VLD measurements was statistically significantly different in VD of 3 mm × 3 mm DCP (p = 0.009), VLD of 3 mm × 3 mm SCP (p = 0.000), and VLD of 3 mm × 3 mm DCP (p = 0.021). No statistically significant differences were found in VD of 3 mm × 3 mm SCP (p = 0.128) or VLD of 3 mm × 3 mm SCP (p = 0.107). As expected, we were able to demonstrate significant differences in quantitative OCTA parameters gathered from the same images using different methods of quantification. Values gathered using different methods are not interchangeable. In scientific studies and in situations where long-term follow-up is necessary, the same device and the same method of quantification should be used to maintain retrospective comparability of measurements.

Inter-reader agreement of 18F-FDG PET/CT for the quantification of carotid artery plaque inflammation.

IntroductionA significant proportion of ischemic strokes are caused by emboli from unstable atherosclerotic carotid artery plaques. Inflammation is a key feature of plaque instability. Positron emission tomography/computed tomography (PET/CT) with 2-deoxy-2-(18F)-fluoro-D-glucose (18F-FDG) is a promising technique to quantify plaque inflammation, but a consensus on the methodology has not been established. High inter-reader agreement is essential if 18F-FDG PET/CT is to be used as a clinical tool for the assessment of unstable plaques and stroke risk.MethodsWe assessed the inter-reader variability of different methods for quantification of 18F-FDG uptake in 43 patients with carotid artery stenosis ≥70%. Two independent readers delineated the plaque and collected maximum standardized uptake value (SUVmax) from all axial PET slices containing the atherosclerotic plaque.ResultsUptake values with and without background correction were calculated and intraclass correlation coefficients were highest for uncorrected uptake values (0.97–0.98) followed by those background corrected by subtraction (0.89–0.94) and lowest for those background corrected by division (0.74–0.79).ConclusionQuantification methods without background correction have the highest inter-reader agreement for 18F-FDG PET of carotid artery plaque inflammation. The use of the single highest uptake value (max SUVmax) from the plaque will facilitate the method’s clinical utility in stroke prevention.

Midfacial Advancement Line—A Comparative Evaluation of a New Measurement Method in Orthognathic Surgery

In planning intraoral quadrangular Le Fort II osteotomy (IQLFIIO), simulation of hard and soft tissue changes will be important at the infraorbital, Le Fort I, and incisor level. The aim of our study was to evaluate a new method for visualization and quantification. Three different methods of quantification were compared: the point-to-point (PTP) measurement, which has been viewed as the reference standard; part comparison analysis (PCA); and a new method, the midfacial advancement line (MFAL) measurement. We performed a measurement comparison study using the Bland-Altman method to measure agreement and enrolled patients with midfacial deficiency and Class III malocclusion who had undergone IQLFIIO. The primary predictor variable was the method of measurement. The primary outcome variable was the amount of midfacial advancement. We also investigated the time required, visualization quality, and interobserver agreement. The sample included 12 subjects with a mean age of 21.6years; 7 patients were male. The PTP and MFAL showed no significant observer dependence. The advancement measured with PTP and MFAL showed no significant differences. However, the advancement measured using MFAL and PCA showed a significant difference. The highest rating of visualization was found for MFAL. The time requirements were similar for all 3 methods. Our results have shown that the MFAL is a suitable method for visualization and quantification of soft and hard tissue changes at all 3 face levels in 1 image. It could be a valuable tool for virtual planning of midfacial advancement surgery.

18F-FDG PET/CT for the quantification of inflammation in large carotid artery plaques

BackgroundThere is currently no consensus on the methodology for quantification of 18F-FDG uptake in inflammation in atherosclerosis. In this study, we explore different methods for quantification of 18F-FDG uptake in carotid atherosclerotic plaques and correlate the uptake values to histological assessments of inflammation. Methods and ResultsForty-four patients with atherosclerotic stenosis ≥70% of the internal carotid artery underwent 18F-FDG PET/CT. Maximum standardized uptake values (SUVmax) from all plaque-containing slices were collected. SUVmax for the single highest and the mean of multiple slices with and without blood background correction (by subtraction (cSUV) or by division (target-to-background ratio (TBR)) were calculated. Following endarterectomy 30 plaques were assessed histologically. The length of the plaques at CT was 6-32 mm. The 18F-FDG uptake in the plaques was 1.15-2.66 for uncorrected SUVs, 1.16-3.19 for TBRs, and 0.20-1.79 for cSUVs. There were significant correlations between the different uptake values (r = 0.57-0.99, P < 0.001). Methods with and without blood background correction showed similar, moderate correlations to the amount of inflammation assessed at histology (r = 0.44-0.59, P < 0.02). ConclusionsIn large stenotic carotid plaques, 18F-FDG uptake reflects the inflammatory status as assessed at histology. Increasing number of PET slices or background correction did not change the correlation.

Fatty acid composition and nutritional profiles of Brycon spp. from central Amazonia by different methods of quantification.

This study evaluated the fatty acid composition and the nutritional profile of Brycon cephalus and Brycon microlepis, fish species from the central Amazon basin, by different methods of quantification. The methods applied were: area normalization (MAN), internal standard (MIS), alternative theoretical (MAT) and alternative experimental (MAE). Significant differences were observed between the methods applied and the species studied. MAN supplied poor information about fatty acids composition and diet formulation, presenting only fatty acid profiles. MIS, MAT and MAE supplied fatty acids composition information on a mass basis. MAT and MAE overestimated results, whereas MIS presented the most accurate results. B. cephalus and B. microlepis showed high contents of approximately 65mgg-1 of n-3 fatty acids. Eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) content totaled, 104.37mg 100g-1 and 117.89mg 100g-1 for B. cephalus and B. microlepis, respectively. The nutritional profile of both fish species showed favorable indices for nutritional quality of the lipid fraction, indicating that both Brycon species are healthy dietary choices.

Application of Gray Level Co-Occurrence Matrix Analysis as a New Method for Enzyme Histochemistry Quantification.

Enzyme histochemistry is a valuable histological method which provides a connection between morphology, activity, and spatial localization of investigated enzymes. Even though the method relies purely on arbitrary evaluations performed by the human eye, it is still wildly accepted and used in histo(patho)logy. Texture analysis emerged as an excellent tool for image quantification of subtle differences reflected in both spatial discrepancies and gray level values of pixels. The current study of texture analysis utilizes the gray-level co-occurrence matrix as a method for quantification of differences between ecto-5'-nucleotidase activities in healthy hippocampal tissue and tissue with marked neurodegeneration. We used the angular second moment, contrast (CON), correlation, inverse difference moment (INV), and entropy for texture analysis and receiver operating characteristic analysis with immunoblot and qualitative assessment of enzyme histochemistry as a validation. Our results strongly argue that co-occurrence matrix analysis could be used for the determination of fine differences in the enzyme activities with the possibility to ascribe those differences to regions or specific cell types. In addition, it emerged that INV and CON are especially useful parameters for this type of enzyme histochemistry analysis. We concluded that texture analysis is a reliable method for quantification of this descriptive technique, thus removing biases and adding it a quantitative dimension.

Measuring pulmonary arterial compliance: mission impossible? Insights from a novel in vivo continuous-flow based experimental model.

Arterial compliance (C) is related to the elasticity, size, and geometrical distribution of arteries. Compliance is a determinant of the load that impedes ventricular ejection. Measuring compliance is difficult, particularly in the pulmonary circulation in which resistive and compliant vessels overlap. Comparing different methods for quantification of compliance to a method that involves a continuous flow might help to identify the optimal method. Pulmonary arterial compliance was computed in six pigs based on the stroke volume to pulse pressure ratio, diastolic decay exponential fitting, area method, and the pulse pressure method (PPM). Compliance measurements were compared to those obtained under continuous flow conditions through a right ventricular bypass (Heartware Inc., Miami Lakes, FL, USA). Compliance was computed for various flows using diastolic decay exponential fitting after an abrupt interruption of the pump. Under the continuous flow conditions, resistance (R) was a decreasing function of the flow, and the fitting to P = e-t/RC yielded a pulmonary time constant (RC) of 2.06 s ( ± 0.48). Compliance was an increasing function of flow. Steady flow inter-method comparisons of compliance under pulsatile flow conditions showed large discrepancies and values (7.23 ± 4.47 mL/mmHg) which were lower than those obtained under continuous flow conditions (10.19 ± 1 0.31 mL/mmHg). Best agreement with steady flow measurements is obtained with the diastolic decay method. Resistance and compliance are both flow-dependent and are inversely related in the pulmonary circulation. The dynamic nature of the pulsatile flow may induce a non-uniformly distributed compliance, with an influence on the methods of measurement.

Correction: Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry

Correction: Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry

Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry.

Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging. Given the importance of monitoring cellular NAD+ and NADH levels, it is crucial to have a trustful method to do so. This protocol has the purpose of describing the NAD+ and NADH extraction from tissues and cells in an efficient and widely applicable assay as well as its graphic and quantitative analysis.

Influenza Virus Hemagglutinin Stalk-Specific Antibodies in Human Serum are a Surrogate Marker for In Vivo Protection in a Serum Transfer Mouse Challenge Model.

ABSTRACTThe immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines.

Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.

Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7396-9) contains supplementary material, which is available to authorized users.

Estimation of sacroiliac joint index in normal subjects of various age groups: comparative evaluation of four different methods of quantification in skeletal scintigraphy

To estimate and compare the sacroiliac joint (SIJ) index in skeletal scintigraphy by four different methods of quantification employed in normal subjects of different age groups. The whole-body skeletal survey of 100 subjects, who underwent skeletal scintigraphy three hours after injection of 99mTc-Methylene Diphosphonate (MDP), were selected for this analysis. The patients having previous history of low back pain, joint pain or any benign bone joint disorders (e.g. ankylosing spondylitis, metabolic bone disease, and osteoarthritis), documented bone lesions or tumors within the pelvis region were excluded from the study. All subjects had normal posterior pelvis view on visual assessment in the respective study. Sacroiliac joint index was calculated by quantitative sacroiliac scintigraphy. In each subject, four different methods of quantification were carried out: 1. irregular region of interest (ROI) method, 2. rectangular ROI method, 3. profile peak counts (PPC) method and 4. profile integrated counts (PIC) method and applied to calculate SIJ index. SIJ indices for left and right sacroiliac joints were calculated by dividing the count for each joint by the count for the sacrum. Results obtained by the four methods were compared statistically. The overall SIJ index was found to range from 1.06 to 1.36 in the study population of 100 subjects encompassing all age groups. There was no significant difference in the estimated SIJ index within each age group obtained by the four different methods employed in this study. The values of SIJ index were as follows: in patients aged 2-20 years - they ranged from 1.22 to 1.36; in patients aged 21-40 years - from 1.07 to 1.19; for patients aged 41-60 years - from 1.08 to 1.19 and in patients aged 61 years and older, SIJ values were slightly lower than in other groups and ranged from 1.06 to 1.13. Methods of selecting a region of interest have no significant effect on the calculation of SIJ index and in healthy subjects its values range between 1.06 and 1.36, depending on the age of the subject. The maximum value was observed in patients aged 2-20 years and minimum values were noted in patients aged 61 and older.

Critical evaluation of quantitative methods for the multi-elemental analysis of ancient glasses using laser ablation inductively coupled plasma mass spectrometry

Compositional analyses of ancient glasses have the potential to unravel their provenance and identify the raw materials employed in their manufacture. During the last few years, the analysis of archaeological glasses has increasingly benefited from the introduction and further development of micro-destructive techniques with trace element capability, such as laser ablation-inductively coupled plasma-mass spectrometry. However, while a variety of analytical standards can be employed for quantitative analyses of modern glass, the lack of dedicated certified reference materials represents a major obstacle in the analysis of archaeological specimens. Heterogeneity and the uncertainty in the characterisation of commercially available multi-elemental standards can also negatively influence the calculation of the response factors required to obtain fully quantitative data. Finally, fractionation and matrix effects can strongly affect the measurement, resulting in the possibility of over- or under-estimating the concentration of the analytes. This paper describes a fully quantitative method used for the investigation and characterization of a large set of Roman and late Iron Age glasses used in the making of Iron Age British beads, the results of which are published elsewhere (Bertini et al., J. Archaeol. Sci., 2011, 38, 2750–2766). In this work, the impact of the aforementioned issues and the different methods of quantification used on the precision (determined as the bias between different replicates), accuracy and repeatability of the measurement are evaluated. Particular emphasis is placed on assessing the influence of laser sampling procedures, integration of the transient signal, data reduction strategies, quantification approaches and the possibility of adapting the method to suit a large range of glass compositions.

Comparison of six quantitative methods for the measurement of bone turnover at the hip and lumbar spine using 18F-fluoride PET-CT

The aim of this study was to evaluate the relationship between different quantification methods used for the measurement of bone plasma clearance (K(i)) using F-PET at the hip and lumbar spine. Twelve healthy postmenopausal women aged 52-71 years were recruited. Each participant underwent 60-min dynamic F-PET scans at the lumbar spine and hip on two separate occasions with an injected activity of 90 and 180 MBq, respectively. Image-derived input functions were obtained at the aorta from the lumbar spine scans. K(i) was evaluated using a three-compartment four-parameter model (K(i-4k)), three-compartment three-parameter model (K(i-3k)), Patlak analysis (K(i-Pat)), spectral analysis (K(i-Spec)) and deconvolution (K(i-Decon)). Standardized uptake values (SUVs) were also measured. The Pearson correlation between K(i-4k) and K(i-3k), K(i-Pat), K(i-Spec), K(i-Decon) and SUV were 0.91, 0.97, 0.94, 0.95 and 0.93, respectively, with a significance of P less than 0.0001. The differences between the correlations measured using Fisher's Z-test were not significant (P>0.05). Bland-Altman analysis showed that the limits of agreement for K(i) measured as the SD of the differences were 0.0082 (25.9%), 0.0062 (11.7%), 0.0098 (20.1%) and 0.0056 (25.5%) ml/min/ml, respectively, and the biases were -0.0081 (-23.8%), -0.0075 (-23.7%), -0.0107 (-29.5%) and -0.0015 (0.8%) ml/min/ml, respectively. All five methods of quantification (K(i-3k), K(i-Pat), K(i-Spec), K(i-Decon) and SUV) strongly correlated with K(i-4k). Although systematic differences of up to 29% were found between K(i-4k) and the other methods (K(i-3k), K(i-Pat), K(i-Spec) and K(i-Decon)), these should not affect the conclusions of clinical studies, provided the methods are applied consistently. However, care should be taken when comparing reports that use different methods of quantification.

Immunohistochemical analysis of vascular density and area in colorectal carcinoma using different markers and comparison with clinicopathologic prognostic factors

Analysis of blood and lymphatic vessel in colorectal cancer is controversial in the literature, possibly due to variations in the methods of analysis. In this study, it was aimed to search for a reliable approach in the quantification of angio- and lymphangiovascular density and area as a prognostic factor and to compare such vessel counts in normal mucosa, adenomas and cancer. A retrospective study was performed on 60 sporadic colorectal cancer, 30 colorectal adenomas, and 10 colorectal non-neoplastic lesions. Archival tissues were submitted to immunohistochemical evaluation using antibodies to CD31, CD34, CD105, VEGF-A, VEGF-C, and D2-40. Microvessel density and total vascular area were determined by computer image analysis and values were compared in the three groups of lesions; the prognostic value of these parameters was evaluated in the group of colorectal cancer. Most markers showed progressive vessel counts from non-neoplastic tissue to carcinoma, both for microvessel density and total vascular area. Only microvessel density determined by CD34 in the central areas of the cancer correlated with recurrence/metastasis (p = 0.04) and survival (p = 0.02). Different methods of quantification (microvessel counting versus estimation of total vascular area), immunohistochemical markers (pan-endothelial marker versus neovessels and lymphatic markers), and areas of analysis (periphery versus inner portions of the lesion) were assessed using image analysis. The results corroborate the increase in vascularization of carcinoma and suggest that microvessel density determined by immunostaining for CD34 in the inner portion of the tumor might represent a prognostically relevant parameter in colorectal cancer.

Analysis of Retina and Erythrocyte Glycerophospholipid Alterations in a Rat Model of Type 1 Diabetes.

An automated tandem mass spectrometry based analysis employing precursor ion and neutral loss scans in a triple quadrupole mass spectrometer has been employed to identify and quantify changes in the abundances of glycerophospholipids extracted from retina and erythrocytes in a rat streptozotocin model of type 1 diabetes, 6 weeks and 36 weeks following induction of diabetes, compared to age matched nondiabetic controls. The utility of an 'internal standard' method compared to an 'internal standard free' method for quantification of differences in the abundances of specific lipid ions was evaluated in both retina and erythrocyte lipid extracts. In retina, equivalent results were obtained by using the internal standard and 'internal standard free' methods for quantification. In erythrocytes, the two methods of analysis yielded significantly different results, suggesting that factors intrinsic to particular sample types may influence the outcome of label-free lipidome quantification approaches.Overall increases (~25% to ~35%) in the abundances of major retina glycerophospholipid classes were demonstrated in rats at 6 weeks of diabetes, relative to control animals. However, at 36 weeks of diabetes, subsequent overall decreases in retina glycerophosphocholine and glycerophosphoethanolamine abundances of 16% and 33%, respectively, were observed. Additionally, retina and erythrocyte glycerophosphocholine lipids at both 6 week and 36 weeks of diabetes exhibited increased incorporation of linoleic acid((18:2n6)) and a decrease in docosahexaenoic acid (DHA((22:6n3))) content. Finally, an approximately 5-fold increase in the abundances of specific glycated glycerophosphoethanolamine (Amadori-GPEtn) lipids were observed in the retina of 36 week diabetic rats, with a corresponding 1.6 fold increase of Amadori-GPEtn lipids in diabetic erythrocytes.

Assessment of in vitro methodologies to determine topical and transdermal delivery of the flavonoid quercetin

To be effective against the oxidative damages induced by UVB irradiation in the skin, the drug needs to release from the formulation in which it was incorporated and reach the skin layers where the ROS are generated. Thus, it is very important the development of a robust and sensitive methodology to extract and quantify in different skin layers the antioxidant agent delivered from topical formulations. Therefore, in the present work suitable methods to extract and quantify quercetin in skin samples and receptor phase after in vitro penetration studies were developed. The results demonstrated that the recovery from two different layers of skin, the SC and [E+D], using two different methods of quantification (DPPH• assay and HPLC, respectively), was 93.8 % when the quercetin spiked dose was 50 µg/mL, 100.4 % when it was 100 µg/mL and 89.9 % for 250 µg/mL and the average recovery of the quercetin extraction from receptor phase when dichloromethane was used as extractor solvent was 96%. These results demonstrate that the described methods have a potential application to in vitro skin penetration studies of quercetin, since it showed to be accurate and sensitive.