Optical Density Ratio Research Articles

Mid-season targeted selective anthelmintic treatment based on flexible weight gain threshold for nematode infection control in dairy calves

The suitability of a single mid-season targeted selective treatment (TST) for gastrointestinal nematodes control, based on flexible average daily weight gain (ADWG) thresholds, was investigated in 23 groups of first grazing season calves. In each group, animals were weighed three times: before turnout, at mid-season and at housing. Just after the first weighing, each group was divided in two homogenous sub-groups in terms of age, breed and weight, and randomly allocated to one of two sub-groups intented for two different mid-season anthelmintic treatment strategies: (1) a treatment of all calves composing the sub-group (whole-group treatment (WT)) or (2) a targeted selective weight gain-based treatment (TST) of the animals showing an individual pre-treatment ADWG inferior to the mean pre-treatment ADWG of the corresponding WT sub-group. Anthelmintic treatment (levamisole 7.5 mg/kg BW) was performed 3 to 4 months after turnout. At housing, two parasitological parameters (the anti-Ostertagia ostertagi antibody level-Ostertagia optical density ratio (ODR) and the pepsinogen level) and a clinical parameter (the breech soiling score) were assessed at individual level in each group. Then, the high exposed groups to gastrointestinal nematode (GIN) were defined as groups for which untreated animals exhibited a mean Ostertagia ODR ⩾0.7 and among these groups, the ones characterized by high abomasal damage due to Ostertagia for which untreated animals exhibited a mean pepsinogen level ⩾2.5 U Tyr were also identified. Among TST sub-groups, the treatment ADWG thresholds varied from 338 to 941 g/day and the percentage of treated animals from 28% to 75%. Pre- and post-treatment ADWG as well as parasitological and clinical parameters measured at housing were similar between TST and WT sub-groups including the 17 high exposed groups to GIN. Within these 17 groups, the treatment allowed to significantly improve post-treatment ADWG compared with untreated animals. In the six high exposed groups showing mean pepsinogen level ⩾2.5 U Tyr, the average effect of treatment on post-treatment ADWG was the highest and estimated up to 14 kg after a grazing duration of 4 months. In contrast, in six other groups showing mean Ostertagia ODR<0.7 in untreated animals, no effect of treatment was seen suggesting an absence of production losses related to a low level of GIN infection. This study highlighted the suitability of a convenient mid-season TST strategy for first grazing season calves, based on the use of flexible thresholds of ADWG, allowing similar growth compared with a whole-group treatment while keeping a GIN population in refugia.

Optical Density of Subretinal Fluid as a Predictive Biomarker for Eyes With Chronic Central Serous Retinopathy Treated With Eplerenone

Purpose: To investigate how the optical density ratio (ODR) of subretinal fluid (SRF) obtained by optical coherence tomography (OCT) in eyes with chronic central serous retinopathy (CSR), defined as symptoms &gt;3 months, treated with eplerenone correlates with treatment response and visual outcomes. Methods: This retrospective, single-center observational study included patients with chronic CSR treated with eplerenone who had at least 6 weeks of follow-up. Eyes with poor-quality OCT scans, shallow SRF precluding sampling of optical density (OD), and macular pathology other than CSR were excluded. Optical density of the vitreous and SRF was measured using ImageJ and used to calculate ODR (ODR = ODSRF/ODVIT). Peak SRF height, foveal SRF height, central macular thickness (CMT), and Snellen visual acuity (VA) were measured. The Mann-Whitney U test was used to compare continuous variables. Pearson correlation coefficient was used to determine the statistical significance of applied linear regressions. Results: Twenty-three eyes of 23 patients met the inclusion criteria. Six (26%) eyes had complete resolution of foveal SRF (resolvers) with eplerenone treatment, and the remainder of patients had worsening or incomplete resolution (nonresolvers). Resolvers had significantly lower baseline logarithm of the minimum angle of resolution (logMAR) VA ( P &lt; .01) than nonresolvers. There was a positive linear correlation between ODR and logMAR VA at baseline ( R2 = 0.208; P = .03) and follow-up ( R2 = 0.206; P = .03). A significant correlation between ODR and percentage change in CMT was found ( R2 = 0.263; P = .01). Linear correlations were not found between ODR and age, peak SRF height, and foveal SRF height (all P &gt; .125). Conclusion: Lower baseline ODR was associated with improved percentage change in CMT and VA in patients treated with eplerenone for chronic CSR. Optical density ratio may represent a novel prognostic biomarker that may help determine which eyes with chronic CSR may respond better to eplerenone.

Apparent prevalence of and risk factors for infection with Ostertagia ostertagi, Fasciola hepatica and Dictyocaulus viviparus in Swiss dairy herds

Infections with helminth parasites can negatively affect performance of dairy cows. Knowledge on infection intensity, spatial distributions and risk factors are key to develop targeted treatment strategies. Canada and most EU countries have conducted large investigations, but respective data for Switzerland were missing. We now performed a bulk tank milk serosurvey for Ostertagia ostertagi, Fasciola hepatica, and Dictyocaulus viviparus on a total of 1036 voluntarily participating dairy herds that were sampled at confinement periods, i.e. in winter 2014/15 or 2015/16, respectively. All samples were analyzed with commercial ELISAs for antibodies (AB) against O. ostertagi and F. hepatica, and those of the first sampling period additionally with an in-house ELISA for AB against D. viviparus. Testing for the latter parasite was not done in the second year of the study, as the sampling period might have missed infections due to the short lived nature of specific antibodies. The possible influence of geographic, climatic, and farm management variables on AB levels were assessed for each parasite using scanning cluster and multiple regression analysis.Overall seroprevalence for O. ostertagi was 95.5% (95% C.I.: 94.0–96.6), with a mean optical density ratio (ODR) of 0.83, for F. hepatica 41.3% (95% C.I.: 38.3–44.4), and for D. viviparus 2.9% (95% C.I.: 1.6–4.7). There were no significant differences between the two sampling periods. For all parasites, significant geographic clusters of higher AB levels could be established. Furthermore, AB levels against all three parasites were positively correlated with each other, indicating either cross-reactions or co-infections. For O. ostertagi, herd size and percentage of pasture in the ration were positively correlated with AB levels. For F. hepatica, altitude above sea level (a.s.l.) positively, and milk production per cow and year was negatively correlated with AB levels.This work provides baseline data for further studies performing in-depth risk factor analysis and investigating management as well as targeted treatment options to control the parasites.

A longitudinal study of gastrointestinal parasites in English dairy farms. Practices and factors associated with first lactation heifer exposure to Ostertagia ostertagi on pasture

The gastrointestinal nematode Ostertagia ostertagi is an important cause of lost production, health, and welfare in cattle. Detailed records were obtained over a 5-yr period (2010-2015) by questionnaires and qualitative interviews to investigate the practices adopted by dairy farmers to control cattle helminth infections and the factors associated with heifer exposure to O. ostertagi on pasture. In total, 1,454 heifers' individual milk samples were collected over a 1-yr period (2014-2015) in 43 dairy farms in England and tested for O. ostertagi antibody by ELISA. Multilevel linear regression models were used to investigate the association between individual milk optical density ratio (ODR) against O. ostertagi and heifer management from birth to time of sampling. Farm and heifer median ODR against O. ostertagi were 0.98 (interquartile range = 0.76-1.02) and 0.64 (interquartile range = 0.42-0.84), respectively. The majority of heifers (88%) received an anthelmintic treatment before sampling in this study. After controlling for the effect of anthelmintic treatments, heifer individual milk ODR against O. ostertagi significantly increased with high stocking rate at first grazing and co-grazing with adult cows before calving. Conversely, heifer individual milk ODR against O. ostertagi significantly decreased when heifers had co-grazed with sheep and pasture grass had frequently been mowed. Overall, these results provide evidence to support targeting grazing management toward limiting the use of anthelmintics in dairy young stock to enable sustainable control of cattle helminth infections in England. However, to be accepted and adopted by farmers, these best practices would need to take into account farmers' perspectives and contextual challenges.

Serological detection of Ascaris suum at fattening pig farms is linked with performance and management indices

The aim of the present study was to determine the association between the presence of Ascaris suum at fattening pig farms, using different serological methods and the percentage of affected livers at slaughter, with performance and management indices. In total, 21 fattening pig farms from the North of Spain were included in the study. Serum samples were collected from pigs at slaughter and analysed for the presence of anti-Ascaris antibodies. For this, two different ELISAs were used. The first was based on the antibody recognition of the A. suum haemoglobin (As-Hb) molecule whereas the second test used the total extract of A. suum lung stage L3. The serological results were subsequently correlated with the percentage of condemned livers at slaughter, management practices and technical performance parameters including average daily gain (ADG) and feed conversion ratio (FCR). According to the data from the slaughterhouse, 12 out of the 21 farms had livers condemned due to liver white spots. A total of 10 farms (48%) had an average optical density ratio (ODr) exceeding the test cutoff when the As-Hb ELISA was used. This number increased to 18 farms (81%) when using the As-Lung-L3 ELISA. The average ODr of the farms on both ELISAs correlated positively with the percentage of affected livers (P<0.01). Only the average ODr values obtained with the As-Lung-L3 ELISA were positively correlated with the FCR (P<0.01). No correlation was found between percentage of affected livers or serology and the ADG. In relation to management practices, farms with greater than or equal to 50% slatted flooring and that applied the ‘all-in/all-out’ flow system showed a lower percentage of liver condemnations (P<0.01), lower average ODr results on the As-Lung-L3 ELISA (P<0.05) and lower FCR (P<0.01) compared with those with less than 50% slatted flooring. This study emphasizes that serology is a promising diagnostic tool for diagnosing ascariasis at fattening pig farms. It also supports earlier findings that the presence of A. suum can have a significant negative impact on farm productivity and that stable infrastructure or management practices can have a considerable impact on the control of this parasite.

Subcutaneous veins depth measurement using diffuse reflectance images.

Intravenous (IV) procedures are often difficult due to the poor visualization of subcutaneous veins. Because existing vein locators lack the ability to assess depth, and also because mis-punctures and poor vascular access remain problematic, we propose an imaging system that employs diffuse reflectance images at three isosbestic wavelengths to measure both the depth and thickness of subcutaneous veins. This paper describes the proposed system as well as proof-of-principle experimental demonstrations. We initially introduce the working principle and structure of the system. All measurements were based on the Monte Carlo (MC) method and accomplished by referring an optical density (OD) ratio to a multi-layer diffuse reflectance model. Results were all validated by comparative ultrasound measurements. Experimental trials included 11 volunteers who were subjected to both ultrasound measurements and the proposed optical process to validate the system's applicability. However, the unreliability of the "thickness" measurement of the vein may be due to the fact that the veins have collapsible walls - so excess pressure by the transducer will give a false thickness.

Serologic Monitoring of Public Health Interventions against Strongyloides stercoralis.

Northwestern Argentina is endemic for soil-transmitted helminths, and annual deworming programs are carried out in prioritized areas. High prevalence of Strongyloides stercoralis was reported in this area; therefore, control programs including ivermectin are being evaluated. The NIE-enzyme linked immunosorbent assay (ELISA) was used for this purpose. In this community trial, two groups of patients, classified according to housing and living conditions were evaluated. Simultaneous with baseline survey, Group 1 was moved to new households with access to improved water and sanitation facilities (W and S), where deworming (MDA, massive drug administration) took place within 1 month; whereas Group 2 received MDA but remained living with unimproved W and S. The mean time interval between baseline and the follow-up was 331 days for Group 1 and 508 for Group 2. Anti-NIE levels were measured for each individual before and after interventions and follow-up optical density (OD) ratios were calculated to quantify the variation. A significant decrease of the anti-NIE levels between baseline and follow-up was observed in both groups. Nonetheless, the number of patients that achieved the cure criteria (OD ratio < 0.6) was higher in Group 1 than Group 2 with values of 72.7% (24/33) and 45.0% (18/40), respectively (P = 0.0197). Our results support the conclusion that a combined intervention including deworming and improvements in life conditions is more effective, in terms of the proportion of subjects cured than deworming alone. Furthermore, we found that NIE-ELISA is a useful test for assessing the response to treatment and to evaluate the outcome of control intervention programs.

The influence of Toll-like recepter 4 activation to the skeletal muscle fibrosis mediated by transforming growth factor-beta1

Objective Obtain the Muscle derived stem cells from the mouse skeletal muscle and observe the influence of Toll-like receptor 4 (TLR4) activation to the skeletal muscle fibrosis mediated by transforming growth factor-beta1. Methods Cell culture: muscle-derived stem cells (MDSCs) from primary cultures of mouse skeletal muscle were isolated and purified by the preplate technique. Then, the MDSCs were divided into 3 average groups (A, B, C) at random. Group A is the control, Group B cultured in vitro with the transforming growth factor-β1 (TGF-β1), Group C cultured in vitro with the TGF-β1 and heat shock protein 60 (Hsp60)respectively for 3 days. At first, the level of Nuclear transcription factors κB (NF-κB)was tested by RT-PCR and Western blotting to make clear if the TLR4 was activated. Then the level of CTGF and collagen I in these three groups were tested by RT-PCR, Western blotting and immunofluorescence. Animal model: 24 adult male mouses (15-18 g) were divided into three groups (D, E, F) of 8 each at random. The saline, rhTGF-β1 solution and rhTGF-β1 with Hsp60 solution were injected into the left gastrocnemius medial head respectively per 7 days for four times. At the 28th day, the left gastrocnemius medial head was removed to test the wet weight of the muscles and Von Geison stain performed to observe the muscular fibrosis of each group. Results Cell culture: RT-PCR show that the relative expression of the NF-κB of group A, B and C were respectively 0.852±0.121, 0.987±0.084 and 1.304±0.171 (P=0.031). The CTGF of group A, B and C were respectively 0.756±0.205, 0.977±0.188, 1.308±0.302 (P=0.022). The collagen I of group A, B and C were respectively 0.767±0.148, 0.998±0.138, 1.261±0.327 (P=0.016). Western blotting show that the optical density ratio of the NF-κB of group A, B and C were respectively 0.784±0.133, 0.887±0.066, 0.994±0.171 (P=0.026). The CTGF of group A, B and C were respectively 0.644±0.125, 0.863±0.109, 0.998±0.512 (P=0.018). The collagen I of group A, B and C were respectively 0.347±0.133, 0.761±0.086, 0.868±0.167 (P=0.045). In response to the Western blotting, immunofluorescence showed that the expression of the CTGF and collagen I of group A, B and C were increased gradually and there was significance of all the tests statistically between each group (P=0.025, P=0.012). Animal model: The mean value of the wet weight of the left gastrocnemius medial head of group D, E and F are respectively (0.015±0.002), (0.014±0.002), (0.012±0.003) g (P=0.037), PD vs. E=0.033, PD vs. F=0.029, PE vs. F=0.016.The Von Geison stain obtained the integrated optical density ratio of D, E and F group were respectively 100.8±10.3, 112.7±11.1 and 131.9±12.5 (P=0.013, PD vs. E=0.041, PD vs. F=0.027, PE vs. F=0.010). Conclusion The activation TLR-4 of the mouse MDSCs can promote the skeletal muscular fibrosis mediated by TGF-β1. Key words: Muscle-derived stem cells; Ttransforming growth factor-β1; Tool-like receptor; Connective tissue growth factor; Fibrosis

Objective Quantification of Anterior Chamber Inflammation: Measuring Cells and Flare by Anterior Segment Optical Coherence Tomography

To assess the ability of swept-source (SS) optical coherence tomography (OCT) of the anterior segment (AS) to measure anterior chamber (AC) inflammation (both flare and cells) objectively. To compare OCT-derived inflammatory indices with standard techniques. Prospective evaluation of a diagnostic test. Patients diagnosed with anterior uveitis (active or inactive) and controls. Participants underwent an AC inflammation evaluation including: clinical cell and flare grading and laser flare photometry (LFP). Uveitis patients were divided into active or inactive uveitis status according to clinical grading. Anterior segment SS-OCT scans were obtained for each participant. Tomographic images were analyzed to count the AC cells, and to calculate to absolute measurements of aqueous signal intensity. The absolute values were compared with the signal measured by the scan outside the eye, generating an optical density ratio (aqueous-to-air relative intensity [ARI] index). Correlations between OCT-derived AC inflammatory indexes and LFP, clinical grading, participant category (active or inactive uveitis, control), age, gender, and central corneal thickness (CCT) were assessed. Correlation between OCT-derived AC inflammatory indexes (ARI index and AC cells on OCT) and standard clinical techniques (LFP, clinical cell grading). Two hundred thirty-seven eyes (70 active uveitis, 97 inactive uveitis, and 70 controls) were included. Anterior chamber cells count on OCT did not differ between inactive uveitis and controls, but was significantly higher in active uveitis compared to the other categories (both P < 0.0001). All groups had different LFP (all P < 0.0001). Active uveitis had significantly higher ARI index compared with inactive uveitis and controls (both P < 0.0001). Interobserver agreement (intraclass correlation coefficient) for ARI index was 0.78. The ARI index correlated positively with age (P= 0.043) and negatively with CCT (P= 0.006). The ARI index correlated with LFP in the active uveitis group (P < 0.0001), but not in the others. Anterior chamber cells on OCT increased among all cell clinical grades (P < 0.0001). The ARI index increased among all flare clinical grades (P < 0.005). Anterior segment SS-OCT could be used for a comprehensive assessment of AC inflammation, providing objective measurements of inflammatory cells and aqueous flare.

Associations between fasciolosis and milk production, and the impact of anthelmintic treatment in dairy herds

Liver fluke is a ubiquitous parasite that causes extensive production losses in cattle and is a zoonosis. The aims of this study were to determine the prevalence of fasciolosis in 178 dairy cattle herds in Styria (federal state of Austria) and its influence on production, to detect the risk factors for infection, and to explore effective strategies in management and control. A questionnaire on farm management, prophylaxis, and therapy was developed and applied. Furthermore, production parameters (milk yield, milk protein content, butter fat content, non-return rate 90, calving to conception interval, service period) were recorded for 2014 and 2015, and a commercial ELISA for detection of Fasciola hepatica antibodies was applied in bulk tank milk in March 2014 and March 2015. Analysis of bulk tank milk samples showed a prevalence of 61.3% in 2014 and 45.5% in 2015. No associations could be found between F. hepatica exposure and farm structure or pasture management. Farms with highly positive (optical density ratio (ODR) ≥ 0.6 and lying above the upper interquartile range) antibody levels had a significantly lower annual milk yield of 438 kg per cow per year (p = 0.045), butterfat content of 0.091% (p = 0.004), and milk protein content of 0.046% (p = 0.024). However, fertility parameters were not significantly associated with liver fluke exposure. Anthelmintic treatment led to significantly lower antibody levels in the subsequent year (p = 0.042) and had a significant influence on protein content in milk (p = 0.003). This study highlighted the importance of fasciolosis in Austria and its influence on milk production and the need for veterinary advice regarding prophylactic measures to reduce economic losses.

Relationship between CHOP/GADD153 and unstable human carotid atherosclerotic plaque

Background and aims: The signaling protein C/EBP homologous protein (CHOP) and corresponding growth-arrest-and-DNA-damage-inducible gene 153 (GADD153) is associated with endoplasmic reticulum stress (ERS), which can lead to apoptosis. Our study aims to elucidate the role of CHOP/GADD153 in unstable atherosclerotic (AS) plaque formation isolated from confounding factors such as diabetes mellitus, primary hyperlipidemia, autoimmune deficiencies/abnormalities, essential hypertension, chronic heart failure, chronic kidney disease, and smoking.Material and methods: We collected carotid artery tissue samples from patients aged 50–80 years-old who received carotid endarterectomies (CEA) at our institution. We obtained fresh AS plaque samples during CEA and preserved the specimens immediately in the operating room with liquid nitrogen. Samples were categorized as stable or unstable AS plaques according to a six-stage histologic classification. CHOP/GADD153 expression was then examined with immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).Results: A total of 32 patients met our inclusion and exclusion criteria, with 24 (75.0%) classified as unstable lesions. The mean optical density ratio normalized to GAPDH for CHOP/GADD153 in stable and unstable groups was 0.357 ± 0.025 and 0.490 ± 0.027, respectively (p < .05). Positive immunostaining of CHOP/GADD153 was found in macrophages and smooth muscle cells of unstable AS plaques with a mean integrated optical density of 0.63 ± 0.03, compared to 0.17 ± 0.05 in the stable group (p < .05).Conclusions: In conclusion, we were able to show significant elevation of CHOP/GADD153 in unstable plaques independent of other confounding factors that induce ERS.

The association of different auto-antibodies against ovarian tissues and gonadotropins and poor ovarian response in intracytoplasmic sperm injection cycles

This study aimed to assess the possible association between ovarian auto-antibodies and poor ovarian response to controlled ovarian hyperstimulation (COH) in patients undergoing intracytoplasmic sperm injection (ICSI) cycles. In total, 42 poor responders and 43 male factor subjects were enrolled in the study and underwent either a standard long gonadotropin-releasing hormone (GnRH) agonist or antagonist protocol. Anti-ovarian, anti-oocyte, anti-zona pellucida (anti-ZP) and anti-gonadotropin antibodies in their sera and follicular fluid (FF) were measured by an enzyme-linked immunosorbent assay technique (ELISA). The mean follicular fluid anti-oocyte antibody [ratio of optical density (OD) sample/OD Control] was significantly higher in poor responders compared to the normal group (2.40 ± 1.55 versus 1.72 ± 0.71, p = 0.012). The linear regression analysis showed an inverse correlation between FF anti-oocyte antibody concentrations and the number of: (i) retrieved oocytes (B = −1.212, r = −0.235, p = 0.030); (ii) mature oocytes (B = −1.042, r = −0.234, p = 0.031); (iii) embryos available (B = −0.713, r = −0.228, p = 0.036); and (iv) good quality embryos (B = −0.369, r = −0.229, p = 0.035). However, there were no significant differences between two groups in terms of FF and serum anti-ovarian, anti-gonadotropins and anti-ZP antibodies. The Pearson correlation analysis on 85 infertile patients showed a positive correlation between age and the levels of FF anti-oocyte antibody (r = 0.276, p = 0.010). This study demonstrated that FF anti-oocyte antibody could be associated with poor response to COH in ICSI cycles.

Dual-wavelength retinal images denoising algorithm for improving the accuracy of oxygen saturation calculation.

Noninvasive measurement of hemoglobin oxygen saturation ( SO 2 ) in retinal vessels is based on spectrophotometry and spectral absorption characteristics of tissue. Retinal images at 570 and 600 nm are simultaneously captured by dual-wavelength retinal oximetry based on fundus camera. SO 2 is finally measured after vessel segmentation, image registration, and calculation of optical density ratio of two images. However, image noise can dramatically affect subsequent image processing and SO 2 calculation accuracy. The aforementioned problem remains to be addressed. The purpose of this study was to improve image quality and SO 2 calculation accuracy by noise analysis and denoising algorithm for dual-wavelength images. First, noise parameters were estimated by mixed Poisson–Gaussian (MPG) noise model. Second, an MPG denoising algorithm which we called variance stabilizing transform (VST) + dual-domain image denoising (DDID) was proposed based on VST and improved dual-domain filter. The results show that VST + DDID is able to effectively remove MPG noise and preserve image edge details. VST + DDID is better than VST + block-matching and three-dimensional filtering, especially in preserving low-contrast details. The following simulation and analysis indicate that MPG noise in the retinal images can lead to erroneously low measurement for SO 2 , and the denoised images can provide more accurate grayscale values for retinal oximetry.

In vivo oximetry of human bulbar conjunctival and episcleral microvasculature using snapshot multispectral imaging

Multispectral imaging (MSI) is a well-established technique for non-invasive oximetry of retinal blood vessels, which has contributed to the understanding of a variety of retinal conditions, including glaucoma, diabetes, vessel occlusion, and retinal auto-regulation. We report the first study to use snapshot multi-spectral imaging (SMSI) for oximetry of the bulbar conjunctival and episcleral microvasculature in the anterior segment of the eye. We report the oxygen dynamics of the bulbar conjunctival and episcleral microvasculature at normoxia and at acute mild hypoxia conditions.A retinal-fundus camera fitted with a custom Image-Replicating Imaging Spectrometer was used to image the bulbar conjunctival and episcleral microvasculature in ten healthy human subjects at normoxia (21% Fraction of Inspired Oxygen [FiO2]) and acute mild hypoxia (15% FiO2) conditions. Eyelid closure was used to control oxygen diffusion between ambient air and the sclera surface. Four subjects were imaged for 30 seconds immediately following eyelid opening. Vessel diameter and Optical Density Ratio (ODR: a direct proxy for oxygen saturation) of vessels was computed automatically. Oximetry capability was validated using a simple phantom that mimicked the scleral vasculature.Acute mild hypoxia resulted in a decrease in blood oxygen saturation (SO2) (i.e. an increase in ODR) when compared with normoxia in both bulbar conjunctival (p < 0.001) and episcleral vessels (p = 0.03). Average episcleral diameter increased from 78.9 ± 8.7 μm (mean ± standard deviation) at normoxia to 97.6 ± 14.3 μm at hypoxia (p = 0.02). Diameters of bulbar conjunctival vessels showed no significant change from 80.1 ± 7.6 μm at normoxia to 80.6 ± 7.0 μm at hypoxia (p = 0.89). When exposed to ambient air, hypoxic bulbar conjunctival vessels rapidly reoxygenated due to oxygen diffusion from ambient air. Reoxygenation occured in an exponential manner, and SO2 reached normoxia baseline levels. The average ½ time to full reoxygenation was 3.4 ± 1.4 s. As a consequence of oxygen diffusion, bulbar conjunctival vessels will be highly oxygenated (i.e. close to 100% SO2) when exposed to ambient air. Episcleral vessels were not observed to undergo any significant oxygen diffusion, instead behaving similarly to pulse oximetry measurements.This is the first study to the image oxygen dynamics of bulbar conjunctival and episcleral microvasculature, and consequently, the first study to directly observe the rapid reoxygenation of hypoxic bulbar conjunctival vessels when exposed to ambient air. Oximetry of bulbar conjunctival vessels could potentially provide insight into conditions where oxygen dynamics of the microvasculature are not fully understood, such as diabetes, sickle-cell diseases, and dry-eye syndrome. Oximetry in the bulbar conjunctival and episcleral microvasculature could be complimentary or alternative to retinal oximetry.

MP44-07 THE USE OF CLARITY-OPTIMIZED LIGHT-SHEET MICROSCOPY (COLM) FOR HIGH RESOLUTION IMAGING OF THE ADULT MOUSE PROSTATE

secondary monoclonal antibodies. This type of measurement is also known as sandwich assay as the analyte is “sandwiched” between two antibodies. The following addition of TMB (Tetramethylbenzidine) was resulting in colour change which intensity was in direct ratio of optical density of the samle and could be read on a spectrophotometer at a wavelength of 650 nm. RESULTS: The following variables were assessed: the mean level of EGF in urine in BPH patients was 14,09 ng/ml (13-32), in urine of men without any pathology and signs of LUTS e 7,86 ng/ml (6,6-9). The mean level of EGF in urine was 80% higher in BPH group in comparison with men without any signs of LUTS due to BPO. We have also found the inverse proportion of Qmean and Qmax and the EGF level in group of BPH patients. CONCLUSIONS: The EGF level in urine could be an important prognostic value in diagnostics and monitoring of patients with LUTS along with standard urodynamic methods and all-known progression risk factors such as age, IPSS, prostate volume, Qmax, PVR and PSA.

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections.

Quality Analysis of DNA from Cord Blood Buffy Coat: The Best Neonatal DNA Source for Epidemiological Studies?

Umbilical cord blood is an economical and easy to obtain source of high-quality neonatal genomic DNA. However, although large numbers of cord blood samples have been collected, information on the yield and quality of the DNA extracted from cord blood is scarce. Moreover, considerable doubt still exists on the utility of the buffy coat instead of whole blood as a DNA source. We compared the sample storage and DNA extraction costs for whole blood, buffy coat, and all-cell pellet. We evaluated three different DNA purification kits and selected the most suitable one to purify 1011 buffy coat samples. We determined the DNA yield and optical density (OD) ratios and analyzed 48 single-nucleotide polymorphisms using time-of-flight mass spectrometry (TOF MS). We also analyzed eight possible preanalytical variables that may correlate with DNA yield or quality. Buffy coat was the most economical and least labor-intensive source for sample storage and DNA extraction. The average yield of genomic DNA from 200 μL of buffy coat sample was 16.01 ± 8.00 μg, which is sufficient for analytic experiments. The mean A260/A280 ratio and the mean A260/A230 ratio were 1.89 ± 0.09 and 1.95 ± 0.66, respectively. More than 99.5% of DNA samples passed the TOF MS test. Only hemolysis showed a strong correlation with OD ratios of DNA, but not with yield. Our findings show that cord blood buffy coat yields high-quality DNA in sufficient quantities to meet the requirements of experiments. Buffy coat was also found to be the most economic, efficient, and stable source of genomic DNA.

A.183 - Gafchromic EBT3 calibration: A new curve type reducing the dependence on films lot

Introduction: The aim of this work is to describe a new form of calibration curve for EBT3 Gafchromic film, using a net optical density ratio, and to compare it with other two types of calibration curves, commonly used so far, using the optical density or the net optical density.

A New Image Analysis Method Based on Morphometric and Fractal Parameters for Rapid Evaluation of In Situ Mammalian Mast Cell Status.

Apart from their effector functions in allergic disorders, tissue-resident mast cells (MC) are gaining recognition as initiators of inflammatory events through their distinctive ability to secrete many bioactive molecules harbored in cytoplasmic granules. Activation triggers mediator release through a regulated exocytosis named degranulation. MC activation is still substantiated by measuring systemic levels of MC-restricted mediators. However, identifying the anatomical location of MC activation is valuable for disease diagnosis. We designed a computer-assisted morphometric method based on image analysis of methylene blue (MB)-stained normal mouse skin tissue sections that quantitates actual in situ MC activation status. We reasoned MC cytoplasm could be viewed as an object featuring unique relative mass values based on activation status. Integrated optical density and area (A) ratios were significantly different between intact and degranulated MC (p<0.001). The examination of fractal characteristics is of translational diagnostic/prognostic value in cancer and readily applied to quantify cytoskeleton morphology and vasculature. Fractal dimension (D), a measure of their comparative space filling capacity and structural density, also differed significantly between intact and degranulated MC (p<0.001). Morphometric analysis provides a reliable and reproducible method for in situ quantification of MC activation status.

Evaluation of Objective Vitritis Grading Method Using Optical Coherence Tomography: Influence of Phakic Status and Previous Vitrectomy

To evaluate a proposed method for objective measurement of vitreous inflammation using a spectral-domain optical coherence tomography (SD OCT) device in a large cohort of uveitis eyes, including pseudophakic eyes and vitrectomized eyes. Retrospective, observational cohort study. One hundred five uveitis eyes (105 patients) with different vitreous haze score grades according to standardized protocols and corresponding SD OCT images (Cirrus HD-OCT; Carl Zeiss Meditec, Dublin, California, USA) were included. Clinical data recorded included phakic status, previous vitreoretinal surgery, and anterior chamber (AC) cells and flare. SD OCT images were analyzed using custom software that provided absolute measurements of vitreous (VIT) and retinal pigment epithelium (RPE) signal intensities, which were compared to generate a relative optical density ratio with arbitrary units (VIT/RPE-relative intensity) and compared to VHS. VIT/RPE-relative intensity showed a significant positive correlation with vitreous haze score (r = 0.535, P < .001) that remained significant after adjusting for factors governing media clarity, such as AC cells, AC flare, and phakic status (R(2)-adjusted = 0.424, P < .001). Significant differences were also observed between the different vitreous haze score groups (P < .001). Preliminary observation did not observe differences in VIT/RPE-relative intensity values between phakic and pseudophakic eyes (0.3522 vs 0.3577, P = .48) and between nonvitrectomized and vitrectomized eyes (0.3540 vs 0.3580, P = .52), overall and respectively for each vitreous haze score subgroup. VIT/RPE-relative intensity values provide objective measurements of vitreous inflammation employing an SD OCT device. Phakic status and previous vitrectomy surgery do not appear to influence these values, although these preliminary findings need validation in future studies.