Performance analysis of automated evaluation of antinuclear antibody indirect immunofluorescent tests in a routine setting.

PURPOSE: Indirect immunofluorescence (IIF) on the human epithelial cell-line HEp-2 (or derivatives) serves as the gold standard in antinuclear antibody (ANA) screening. IIF, and its evaluation, is a labor-intensive method, making ANA testing a major challenge for present clinical laboratories. Nowadays, several automated ANA pattern recognition systems are on the market. In the current study, the EUROPattern Suite is evaluated for its use in daily practice in a routine setting. METHODS: A total of 1033 consecutive routine samples was used to screen for ANA. Results (positive/negative ANA screening, pattern identification and titer) were compared between software-generated results (EUROPattern) and visual interpretation (observer) of automatically acquired digital images. RESULTS: Considering the visual interpretation as reference, a relative sensitivity of 99.3% and a relative specificity of 88.9% were obtained for negative and positive discrimination by the software (EPa). A good agreement between visual and software-based interpretation was observed with respect to pattern recognition (mean kappa: for 7 patterns: 0.7). Interestingly, EPa software distinguished more patterns per positive sample than the observer (on average 1.5 and 1.2, respectively). Finally, a concordance of 99.3% was observed within the range of 1 titer step difference between EPa and observer. CONCLUSIONS: The ANA IIF results reported by the EPa software are in very good agreement with the results reported by the observer with respect to being negative/positive, pattern recognition and titer, making automated ANA IIF evaluation an objective and time-efficient tool for routine testing.

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

BACKGROUND: Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. METHODS: We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. RESULTS: Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. CONCLUSIONS: Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Effects of herbicide-treated host plants on the development of Mamestra brassicae L. caterpillars.

Herbicides are widely used pesticides that affect plants by changing their chemistry. In doing so, herbicides might also influence the quality of plants as food for herbivores. To study the effects of herbicides on host plant quality, 3 plant species (Plantago lanceolata L., P. major L., and Ranunculus acris L.) were treated with sublethal rates of either a sulfonylurea (Atlantis WG, Bayer CropScience) or a glyphosate (Roundup LB Plus, Monsanto) herbicide, and the development of caterpillars of the cabbage moth Mamestra brassicae L. that fed on these plants was observed. Of the 6 tested plant-herbicide combinations, 1 combination (R. acris + sulfonylurea herbicide) resulted in significantly lower caterpillar weight, increased time to pupation, and increased overall development time compared with larvae that were fed unsprayed plants. These results might be caused by a lower nutritional value of these host plants or increased concentrations of secondary metabolites that are involved in plant defense. The results of the present and other studies suggest potential risks to herbivores that feed on host plants treated with sublethal rates of herbicides. However, as the effects of herbicides on host plant quality appear to be species-specific and as there are numerous plant-herbicide-herbivore relationships in agricultural landscapes, a general reduction in herbicide contamination of nontarget habitats (e.g., field margins) might mitigate the negative effects of herbicides on host plant quality.

Characterization of field margins in intensified agro-ecosystems-why narrow margins should matter in terrestrial pesticide risk assessment and management.

Field margins are important seminatural habitats in agro-ecosystems, but they can be negatively affected by pesticide inputs via direct overspray and spray drift. In Germany, risk mitigation measures (like buffer zones) to reduce pesticide inputs in terrestrial noncrop habitats do not have to be put in place by farmers next to narrow field margins (<3 m width). Because data on structure, size, and width of field margins are scarce, we identified field margins in 2 German agricultural landscapes (Rhineland-Palatinate [RLP], Brandenburg [BB]; 4000 ha each) using digital orthophotos and geographical information systems. In RLP, most of the field margins were less than 3 m wide (85% of margin length and 65% of the margin area), whereas in BB narrow field margins account for 45% of the margin length and 17% of the margin area. Hedgerows were only occasionally recorded. Hence, narrow grassy field margins can represent a large part of the available seminatural habitats adjoining agricultural sites and potentially act as corridors between further habitat patches. For this reason, these margins should be protected from pesticide inputs, at least in landscapes under intensive agricultural use. Field margins are also the main, so-called nontarget habitat protected by the terrestrial risk assessment for plants and arthropods. With many (narrow) margins not considered relevant for risk management, the current practice for protecting the biodiversity from negative effects of pesticides seems questionable. More data on field margin constitution in Germany and other European countries is necessary to critically assess the current practice of pesticide risk assessment and management on a larger scale.

Automated indirect immunofluorescence evaluation of antinuclear autoantibodies on HEp-2 cells.

Indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells is considered as the gold standard screening method for the detection of antinuclear autoantibodies (ANA). However, in terms of automation and standardization, it has not been able to keep pace with most other analytical techniques used in diagnostic laboratories. Although there are already some automation solutions for IIF incubation in the market, the automation of result evaluation is still in its infancy. Therefore, the EUROPattern Suite has been developed as a comprehensive automated processing and interpretation system for standardized and efficient ANA detection by HEp-2 cell-based IIF. In this study, the automated pattern recognition was compared to conventional visual interpretation in a total of 351 sera. In the discrimination of positive from negative samples, concordant results between visual and automated evaluation were obtained for 349 sera (99.4%, kappa = 0.984). The system missed out none of the 272 antibody-positive samples and identified 77 out of 79 visually negative samples (analytical sensitivity/specificity: 100%/97.5%). Moreover, 94.0% of all main antibody patterns were recognized correctly by the software. Owing to its performance characteristics, EUROPattern enables fast, objective, and economic IIF ANA analysis and has the potential to reduce intra- and interlaboratory variability.

Age-related differences in performance and stimulus processing in dual task situation.

Competition for limited processing resources is most critical in dual-tasks which incorporate cognitive and motor demands. Performance is usually diminished with increasing age in such tasks. This decline is relevant for activities in real life like driving. In the present study we aim to examine if there are age-related differences in stimulus processing in a dual-task and if these differences have an impact on performance of a driving-like tracking task. Young and older participants performed a dual-task consisting of a tracking task and a visual attention task. Alongside, the EEG was recorded for calculating the P300 (P3) of the event-related potential as a reflection of controlled stimulus processing. In the visual attention task older vs. young participants showed more misses and false alarms. For young participants the P3 shows an expected pattern of higher amplitudes for relevant compared to irrelevant stimuli. This was not found for the older participants. A general age-related decline in tracking performance was relatively more pronounced if a secondary motor response was required in the visual attention task and also after irrelevant stimuli. The results suggest that older compared to young participants had greater difficulties to differentiate between relevant and irrelevant stimuli in a dual-task situation as they probably apply comparable attentional resources to all stimuli. This may also explain the higher error rates. These results have important implications for the understanding of age-related stimulus processing in dual and multi task situations in real life as for instance driving.

Preparatory processes and compensatory effort in older and younger participants in a driving-like dual task.

OBJECTIVE: The nature of increased-age-related dual-task interference was examined during a driving-like dual task in the laboratory. BACKGROUND: Previous research revealed age-related deficits in dual tasks especially when cognitive and motor demands are involved. The specific contributions of sensory input, working memory demands, and/or coordination of motor responses to dual-task interference are not clear and should be clarified in the present study. METHOD: Younger and older participants performed a driving-like tracking task and a visually cued attention task within a dual task. Behavioral and electrophysiological data were recorded during task performance. RESULTS: Overall tracking performance was lower for the older versus younger participants. This age-related decline was particularly pronounced in the time interval after the stimulus when the attention task demanded a motor response. In contrast, older participants tracked relatively better than the younger participants in the time interval preceding the stimulus. In the attention task, the older versus younger participants showed increased responses times and rates of false alarms and misses, suggesting a deficit in retaining the context in the cue-stimulus interval. The electroencephalogram data suggest that the older participants invested more resources than the younger participants in dual-task management during the cue-stimulus interval. CONCLUSION: Evidence was found for increased motor interference and a deficient context processing as well as for an increased investment of processing resources in the older compared with the younger group. APPLICATION: The results suggest that in-vehicle information systems for older drivers should be designed to support cue maintenance and that simultaneous motor demands should not be required.

Annexin and survivin in locally advanced rectal cancer: indicators of resistance to preoperative chemoradiotherapy?

BACKGROUND: The aim of preoperative chemoradiotherapy is improvement of local control in patients with locally advanced rectal cancer (LARC). Recent studies have shown that annexin and survivin are involved in the resistance capability of tumours. We sought to determine whether survivin, annexin A4 or annexin A5 expression predict resistance to preoperative chemoradiotherapy. MATERIAL AND METHODS: Biopsies of tumour and normal rectal tissue were taken from 38 patients with LARC (cT3/4Nx or Tx/N+) before the start of chemoradiotherapy and during surgery. mRNA expression of annexin A4/A5 and survivin was measured by real-time polymerase chain reaction (RT-PCR) and correlated with down-staging and progression-free survival (PFS). RESULTS: Significantly higher mRNA levels of survivin, and annexin A4/A5 were detected in untreated tumour compared with normal mucosa. After chemoradiotherapy, this difference disappeared for survivin and annexin A4. Annexin A5 expression in the tumour increased during chemoradiotherapy. No correlation between the mRNA levels of survivin, annexin A4/A5 and tumour down-staging or PFS was noticed. CONCLUSIONS: In the present analysis of 38 patients with LARC undergoing neoadjuvant chemoradiotherapy, the expression levels of survivin and annexin A4 and A5 did not correlate with down-staging. Moreover, with regard to PFS, none of these markers was found to be prognostically relevant.

Aversive associative conditioning of prepulses in a startle inhibition paradigm.

Prepulse inhibition of startle (PPI) represents an automatic mechanism that reflects sensorimotor gating and early attention processes. PPI neither is the consequence of conscious behavioral modulation nor does it depend on learning and conditioning. However, pairing of weak tones and aversive startle stimuli during PPI testing may induce associative learning. Thus, in the present study (n=60) we tested whether prepulses may be subject to aversive conditioning. Eyeblink EMG and electrodermal responses to intense (100 dB) acoustic stimuli, presented either alone or preceded by weak tones (prepulses, 50 ms, 70 dB, SOA=120 ms), were measured. We found that after strong contingent pairing of weak tones with startle stimuli (PPI paradigm) intense versions of these tones induced significantly larger eyeblink and skin conductance responses than did never paired control tones. We conclude that during PPI testing, prepulses may be subject to aversive conditioning.

Cardiovascular reactivity to mental stress is not affected by alpha2-adrenoreceptor activation or inhibition.

RATIONALE: It has been postulated that cardiovascular reactivity to mental stress varies with tonic central sympathetic nervous system activity, but pharmacological evidence is missing. OBJECTIVE: To test whether modulation of central sympathetic nervous system activity by alpha2-adrenergic agonism and antagonism affects cardiovascular reactivity to mental stress. MATERIALS AND METHODS: On three five-stepped dose/concentration-response study days, 12 healthy male volunteers received intravenous infusions of dexmedetomidine (alpha2-agonist, target plasma concentrations: 0.04-0.32 ng/ml), yohimbine (alpha2-antagonist, doses: 0.016-0.125 mg/kg), and placebo, respectively. During each dose step, subjects performed a 5-Choice Reaction Time Task (CRTT) and a Paced Auditory Serial Addition Task (PASAT) to induce moderate mental stress. Prestress baseline, as well as stress-induced responses of heart rate, and noninvasive finger arterial blood pressure (Finapres) were assessed. RESULTS: Prestress baseline heart rate and blood pressure decreased with increasing doses of dexmedetomidine and increased with increasing doses of yohimbine. However, dexmedetomidine and yohimbine did not affect stress-induced heart-rate and blood-pressure changes. CONCLUSIONS: Cardiovascular reactivity to mental stress is not related to pharmacologically manipulated tonic central sympathetic nervous system activity by alpha2-adrenergic agonists and antagonists. These results do not support the assumption that cardiovascular reactivity is an index of tonic central sympathetic nervous system activity.