Definitions and major prerequisites of direct and indirect approaches for estimating reference limits.

Reference intervals are established either by direct or indirect approaches. Whereas the definition of direct is well established, the definition of indirect is still a matter of debate. In this paper, a general definition that covers all indirect models presently in use is proposed. With the upcoming popularity of indirect models, it has become evident that further partitioning strategies are required to minimize the risk of patients' false classifications. With indirect methods, such partitions are much easier to execute than with direct methods. The authors believe that the future of reference interval estimation belongs to indirect models with big data pools either from one laboratory or combined from several regional centres (if necessary). Independent of the approach applied, the quality assurance of the pre-analytical and analytical phase, considering biological variables and other confounding factors, is essential.

Problems with estimating reference change values (critical differences).

The concept of reference change values (RCVs) for diagnosis and monitoring of diseases has become well established. Several models habe been developed, e. g. one assuming a normal distribution and another one for a log-normal distribution. RCV values calculated for some measurands with both models are compared with each other and led to similar results. A few examples led to RCV values which are not plausible for diagnostic purposes. Although statistical concepts of RCV values are well established, their clinical relevance remains questionable at least for some measurands. Studies with clinicians are required whether RCVs are of practical usefulness.

The importance of correct stratifications when comparing directly and indirectly estimated reference intervals.

OBJECTIVES: There are generally two major reasons for the comparison of reference intervals (RIs): when externally determined RIs (from the literature or provided by a manufacturer) are compared with presently used intra-laboratory RIs and when indirectly estimated RIs are compared with directly established RIs. Discrepancies within these comparisons may occur for two reasons: 1. the pre-analytical and/or analytical conditions do not agree and/or 2. biological variables influencing the establishment of RIs have not been considered adequately. If directly and indirectly estimated reference intervals (RIs) are compared with each other, they very often agree. Sometimes, however, a comparison may differ, with the reason for any discrepancy not being further studied. A major reason for differences in the comparison of RIs is that the requirement for stratification has been neglected. METHODS: The present report outlines the consequences to RI comparison if stratification is neglected during RI determination with the main variables affecting RIs being sex and age. Alanine aminotransferase was chosen as an example in which the RIs depend on both these factors. RESULTS: Both direct and indirect approaches lead to erroneous RIs if stratification for variables which are known to affect the estimation of RIs is not performed adequately. However, failing to include a required stratification in procedures for directly determined RIs affects the outcome in a different way to indirectly determined RIs. CONCLUSIONS: The resulting difference between direct and indirect RIs is often misinterpreted as an incorrect RI estimation of the indirect method.

A new indirect estimation of reference intervals: truncated minimum chi-square (TMC) approach.

All known direct and indirect approaches for the estimation of reference intervals (RIs) have difficulties in processing very skewed data with a high percentage of values at or below the detection limit. A new model for the indirect estimation of RIs is proposed, which can be applied even to extremely skewed data distributions with a relatively high percentage of data at or below the detection limit. Furthermore, it fits better to some simulated data files than other indirect methods. The approach starts with a quantile-quantile plot providing preliminary estimates for the parameters (λ, µ, σ) of the assumed power normal distribution. These are iteratively refined by a truncated minimum chi-square (TMC) estimation. The finally estimated parameters are used to calculate the 95% reference interval. Confidence intervals for the interval limits are calculated by the asymptotic formula for quantiles, and tolerance limits are determined via bootstrapping. If age intervals are given, the procedure is applied per age interval and a spline function describes the age dependency of the reference limits by a continuous function. The approach can be performed in the statistical package R and on the Excel platform.

Serum Levels of MicroRNA-371a-3p (M371 Test) as a New Biomarker of Testicular Germ Cell Tumors: Results of a Prospective Multicentric Study.

PURPOSE: Previous studies suggested that serum levels of microRNA (miR)-371a-3p (so-called M371 test) have a much higher sensitivity and specificity than the classic markers of testicular germ cell tumors (GCTs) and are applicable toward both seminoma and nonseminoma. We sought to confirm the usefulness of this test as a novel biomarker for GCT. PATIENTS AND METHODS: In a prospective, multicentric study, serum samples of 616 patients with testicular GCTs and 258 male controls were examined for serum levels of miRNA-371a-3p (miR levels) by quantitative polymerase chain reaction. The GCT population encompassed 359 patients with seminoma and 257 with nonseminoma; 371 had clinical stage I disease, 201 had systemic disease, and 46 had relapses. Paired measurements before and after orchiectomy were performed in 424 patients; 118 with systemic disease had serial measurements during treatment. miR levels were compared with those of ß-human chorionic gonadotropin, α-fetoprotein, and lactate dehydrogenase. RESULTS: For the primary diagnosis of GCT, the M371 test showed a sensitivity of 90.1%, a specificity of 94.0%, an area under the curve of 0.966 upon receiver operating characteristic analysis, and a positive predictive value of 97.2%. α-Fetoprotein, ß-human chorionic gonadotropin, and lactate dehydrogenase had sensitivities of less than 50% in seminoma and slightly higher sensitivities in nonseminomas. miR levels were significantly associated with clinical stage, primary tumor size, and response to treatment. Relapses had elevated miR levels that subsequently dropped to normal upon remission. Teratoma did not express miR-371a-3p. CONCLUSION: The M371 test outperforms the classic markers of GCT with both a sensitivity and a specificity greater than 90%. All histologic subgroups, except teratoma, express this marker. The test could be considered for clinical implementation after further validation.

Challenges and Perspectives in Bridging In- and Outpatient Sectors: The Implementation of Two Alternative Models of Care and Their Effect on the Average Length of Stay.

New models of care aimed at reinforcing the outpatient sector have been introduced in Germany over the last few years. Initially, a subscription-based model ("integrated care") was introduced in 2012 in the Immanuel Klinik Rüdersdorf, wherein patients had to actively subscribe to the integrated care program. This integrated care model was replaced after 2 years by a subscription-free "model project," in which all patients insured by the contracting insurance company took part in the program. Data showed that the introduction of the integrated care program in the inpatient setting led to an increase of the average length of stay in this group. The switch to the model project corrected this unwanted effect but failed in significantly decreasing the average length of stay when compared to standard care. However, both the integrated care program and model project succeeded in reducing the length of stay in the day care setting. When adjusting for the sex and diagnosis proportions of each year, it was shown that diagnosis strongly influenced the average length of stay in both settings, whereas sex only slightly influenced the duration of stay in the inpatient setting. Thus, in spite of strong financial and clinical incentives, the introduction of the model project couldn't fulfill its primary purpose of shifting resources from the inpatient to the outpatient setting in the initial years. Possible explanations, including struggle against long-established traditions and reluctance to change, are discussed.

Reply to the EFSA (2016) on the relevance of recent publications (Hofmann et al. 2014, 2016) on environmental risk assessment and management of Bt-maize events (MON810, Bt11 and 1507).

In this commentary, we respond to a report of the EFSA GMO Panel (EFSA EFSA Supp Publ, 1) that criticises the outcomes of two studies published in this journal (Hofmann et al. Environ Sci Eur 26: 24, 2; Environ Sci Eur 28: 14, 3). Both publications relate to the environmental risk assessment and management of Bt-maize, including maize events MON810, Bt11 and maize 1507. The results of Hofmann et al. (Environ Sci Eur 26: 24, 2), using standardised pollen mass filter deposition measurements, indicated that the EFSA Panel model had underestimated pollen deposition and, hence, exposure of non-target organisms to Bt-maize pollen. The results implied a need for safety buffer distances in the kilometre range for protected nature reserve areas instead of the 20-30 m range recommended by the EFSA Panel. As a result, the EFSA Panel revised their model (EFSA EFSA J 13: 4127, 4), adopting the slope of the empirical data from Hofmann et al. The intercept, however, was substantially reduced to less than 1% at one point by introducing further assumptions based on the estimates of mainly panel members, citing possible 'uncertainty'. Hofmann et al. (Environ Sci Eur 28: 14, 3) published extensive empirical data regarding pollen deposition on leaves. These results were part of a larger 3-year study involving detailed measurements of pollen release, dispersal and deposition over the maize flowering period. The data collected in situ confirmed the previous predictions of Hofmann et al. (Environ Sci Eur 26: 24, 2). Mean levels and observed variability of pollen deposition on maize and four lepidopteran host plants exceeded the assumptions and disagreed with the conclusions of the EFSA Panel. The EFSA Panel reacted in a report (EFSA EFSA Supp Publ, 1) criticising the methods and outcomes of the two published studies of Hofmann et al. while reaffirming their original recommendations. We respond here point-by-point, showing that the critique is not justified. Based on our results on Urtica leaf pollen density, we confirm the need for specific environmental impact assessments for Bt-maize cultivation with respect to protected habitats within isolation buffer distances in the kilometre range.

Critical comments to a recent EFLM recommendation for the review of reference intervals.

In a recent EFLM recommendation on reference intervals by Henny et al., the direct approach for determining reference intervals was proposed as the only presently accepted "gold" standard. Some essential drawbacks of the direct approach were not sufficiently emphasized, such as unacceptably wide confidence limits due to the limited number of observations claimed and the practical usability for only a limited age range. Indirect procedures avoid these disadvantages of the direct approach. Furthermore, indirect approaches are well suited for reference limits with large variations during lifetime and for common reference limits.

Diseases of dab (Limanda limanda): Analysis and assessment of data on externally visible diseases, macroscopic liver neoplasms and liver histopathology in the North Sea, Baltic Sea and off Iceland.

In the framework of the ICON project (Integrated Assessment of Contaminant Impacts on the North Sea), common dab (Limanda limanda) from seven offshore sampling areas in the North Sea, Icelandic waters and the western Baltic Sea were examined in 2008 for the presence of externally visible diseases and parasites (EVD), macroscopic liver neoplasms (tumours) (MLN) and histopathological liver lesions (LH). Methodologies applied followed standardised ICES and BEQUALM protocols. The EDV results revealed pronounced spatial variation, with dab from the central and northern North Sea sampling areas showing the highest disease prevalence. MLN were recorded only in North Sea dab from the German Bight, Firth of Forth and Ekofisk at a low prevalence. LH results revealed a dominant prevalence of non-specific, mostly inflammatory, lesions and a low prevalence of early toxicopathic non-neoplastic lesions, tumour pre-stages (foci of cellular alteration) and liver tumours. For the analysis and assessment of spatial variation of EVD, a Fish Disease Index (FDI) was calculated for individual dab, summarising data on the presence/absence of EDV, their severity grades, effects on the host and compensating for effects of length, sex and season. FDI data confirmed that the health status of North Sea dab from the offshore areas Dogger Bank, Ekofisk and Firth of Forth was significantly worse than in dab from the German Bight, Icelandic areas and the western Baltic Sea. An assessment of the disease data following ICES/OSPAR criteria was accomplished by applying established numeric background (BAC) and ecological assessment criteria (EAC) for EDV, MLN and LH. The combined assessment of the three disease categories indicated that health effects classified as unacceptable were rare and mainly affected dab from the North Sea. Based on the findings of the present study, it is recommended to monitor wild fish diseases in the context of assessing the impact of hazardous substances and other stressors on the marine environment. The Fish Disease Index (FDI) is regarded as a strong tool for disease data analysis and assessment, suitable as ecosystem health indicator.

Integrated indicator framework and methodology for monitoring and assessment of hazardous substances and their effects in the marine environment.

Many maritime countries in Europe have implemented marine environmental monitoring programmes which include the measurement of chemical contaminants and related biological effects. How best to integrate data obtained in these two types of monitoring into meaningful assessments has been the subject of recent efforts by the International Council for Exploration of the Sea (ICES) Expert Groups. Work within these groups has concentrated on defining a core set of chemical and biological endpoints that can be used across maritime areas, defining confounding factors, supporting parameters and protocols for measurement. The framework comprised markers for concentrations of, exposure to and effects from, contaminants. Most importantly, assessment criteria for biological effect measurements have been set and the framework suggests how these measurements can be used in an integrated manner alongside contaminant measurements in biota, sediments and potentially water. Output from this process resulted in OSPAR Commission (www.ospar.org) guidelines that were adopted in 2012 on a trial basis for a period of 3 years. The developed assessment framework can furthermore provide a suitable approach for the assessment of Good Environmental Status (GES) for Descriptor 8 of the European Union (EU) Marine Strategy Framework Directive (MSFD).

Mercury species in dab (Limanda limanda) from the North Sea, Baltic Sea and Icelandic waters in relation to host-specific variables.

In the framework of the ICON project (Integrated Assessment of Contaminant Impacts on the North Sea), muscle tissue from a total of 135 common dab (Limanda limanda) (20-28 cm total length) was collected in seven offshore sampling areas in the North Sea, at Iceland and in the Baltic Sea during Aug/Sept and December 2008 for a chemical mercury speciation analysis by means of gas chromatography and detection by cold vapour atomic fluorescence spectroscopy (GC-CVAFS). There was a highly significant correlation between concentrations of methylmercury (MeHg+) and inorganic mercury (Hg2+) in individual fish, and the mean ratio of MeHg+ compared to Σ Hg (MeHg+ + Hg2+) was 94.0%. The results revealed statistically significant differences in concentrations of MeHg+ and Hg2+, respectively, between sampling areas. Mean concentrations in the German Bight (North Sea), in Icelandic waters and in Mecklenburg Bight (Baltic Sea) were low (MeHg+: 0.023-0.036; Hg2+: 0.001-0.002 mg/kg wet weight), while concentrations in dab from the Dogger Bank, Firth of Forth and the vicinity of the Ekofisk oil field (all North Sea) were significantly higher (MeHg+: 0.059-0.101; Hg2+: 0.003-0.004 mg/kg wet weight). Statistical correlation analysis on effects of host-specific factors revealed that neither length, weight, age, sex nor condition factor showed a significant relationship with Hg concentrations. However, Hg concentrations were significantly correlated with the Fish Disease Index (FDI), indicating a relationship between Hg concentrations and the health status of dab. Multiple linear regression analysis aiming to find factors affecting Hg concentrations revealed that only the sampling area had a highly significant main effect on Hg concentrations, and in some cases, additionally the condition factor contributed significantly to the final model. From the results, it cannot be excluded that elevated Hg concentration recorded in dab were linked to discharges from offshore oil and gas installations and that Hg affected the health status of dab.

Resources of dark skies in German climatic health resorts.

Illumination of nocturnal environments is increasing steadily worldwide. While there are some benefits for mankind, light at night affects animals, plants, and human health by blurring the natural distinction between day and night. International regulations exist to protect the environment for the maintenance of human health but nocturnal darkness is not considered. In Germany, cities and communities labeled as Climatic Health Resorts provide for high standards in air quality. However, their degree of nocturnal darkness is unexplored so far. In our study, we examined the degree of nocturnal darkness in German Climatic Health Resorts by two datasets based on georeferenced remote sensing data. The majority of Climatic Health Resorts (93.1 %) are able to offer a relative respite (≥ 20 mag/arcsec2) from a degraded nocturnal environment, while only 3.4 % are able to offer a dark, if by no means pristine, night environment (≥ 21 mag/arcsec2). Climatic Health Resorts emit less light as well as are less affected by night sky brightness compared to the average of non-classified communities. In combination with daytime requirements, the resorts provide conditions for a more distinct day-and-night-cycle than non-classified communities.

Erratum to: Reply to the EFSA (2016) on the relevance of recent publications (Hofmann et al. 2014, 2016) on environmental risk assessment and management of Bt-maize events (MON810, Bt11 and 1507).

[This corrects the article DOI: 10.1186/s12302-017-0106-0.].

Serum Levels of MicroRNA miR-371a-3p: A Sensitive and Specific New Biomarker for Germ Cell Tumours.

BACKGROUND: Clinical management of germ cell tumours (GCTs) relies on monitoring of serum tumour markers. However, the markers α-fetoprotein (AFP), the ß-subunit of human chorionic gonadotropin (bHCG), and lactate dehydrogenase (LDH) are expressed in <60% of GCT cases. OBJECTIVE: To test the utility of the microRNAs (miRNAs) miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p as sensitive and specific GCT serum biomarkers. DESIGN, SETTING, AND PARTICIPANTS: Serum levels of miRNAs were measured in 166 consecutive patients with GCT before and after treatment and in 106 male controls. In the first 50 consecutive patients, all four miRNAs were measured. In the main study, only the most sensitive miRNA was further analysed. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The specificity and sensitivity of the four miRNAs were studied using receiver operating characteristic curves. miRNA sensitivities were compared to those of classical markers. Statistical cross-comparisons of miRNA levels for GCT subgroups and controls were performed at various time points during treatment. RESULTS AND LIMITATIONS: Overall, miR-371a-3p performed best, with 88.7% sensitivity (95% confidence interval [CI] 82.5-93.3%) and 93.4% specificity (95% CI 86.9-97.3%) and an area under the curve of 0.94, outperforming AFP, bHCG, and LDH (combined sensitivity 50%). According to Kernel density estimation, the sensitivity and specificity were 86.3% and 92.5%, respectively. miR-371a-3p levels dropped to normal after completion of treatment. The miRNA levels correlated with treatment failure and relapse. Teratoma did not express miR-371a-3p. CONCLUSIONS: The miRNA miR-371a-3p is a specific and sensitive novel serum GCT biomarker that accurately correlates with disease activity. Validation of this test in a large-scale prospective study is needed. PATIENT SUMMARY: miR-371a-3p is a novel serum marker for germ cell tumours that is expressed by 88.7% of patients and thus is far more sensitive and specific than classical serum markers. It correlates with tumour burden and treatment results. Validation in a large patient cohort is needed.

Accumulation and variability of maize pollen deposition on leaves of European Lepidoptera host plants and relation to release rates and deposition determined by standardised technical sampling.

BACKGROUND: Risk assessment for GMOs such as Bt maize requires detailed data concerning pollen deposition onto non-target host-plant leaves. A field study of pollen on lepidopteran host-plant leaves was therefore undertaken in 2009-2012 in Germany. During the maize flowering period, we used in situ microscopy at a spatial resolution adequate to monitor the feeding behaviour of butterfly larvae. The plant-specific pollen deposition data were supplemented with standardised measurements of pollen release rates and deposition obtained by volumetric pollen monitors and passive samplers. RESULTS: In 2010, we made 5377 measurements of maize pollen deposited onto leaves of maize, nettle, goosefoot, sorrel and blackberry. Overall mean leaf deposition during the flowering period ranged from 54 to 478 n/cm2 (grains/cm2) depending on plant species and site, while daily mean leaf deposition values were as high as 2710 n/cm2. Maximum single leaf-deposition values reached up to 103,000 n/cm2, with a 95 % confidence-limit upper boundary of 11,716 n/cm2. CONCLUSIONS: Daily means and variation of single values uncovered by our detailed measurements are considerably higher than previously assumed. The recorded levels are more than a single degree of magnitude larger than actual EU expert risk assessment assumptions. Because variation and total aggregation of deposited pollen on leaves have been previously underestimated, lepidopteran larvae have actually been subjected to higher and more variable exposure. Higher risks to these organisms must consequently be assumed. Our results imply that risk assessments related to the effects of Bt maize exposure under both realistic cultivation conditions and worst-case scenarios must be revised. Under common cultivation conditions, isolation buffer distances in the kilometre range are recommended rather than the 20-30 m distance defined by the EFSA.

Myokardszintigraphie - eine Methode zur Erfassung der kardialen Komorbidität bei Psoriasis-Patienten?

HINTERGRUND: Kardiale Komorbiditäten bei Patienten mit Psoriasis stehen seit Jahren im Fokus. Ziel dieser Arbeit war es, im Rahmen einer Pilotstudie die Myokardszintigraphie als mögliche Früherkennungsmethode zu evaluieren. PATIENTEN UND METHODIK: Es wurden bei insgesamt 50 kardial asymptomatischen Patienten mit einer Psoriasis der Haut verschiedene Begleiterkrankungen erfasst. Dabei kam zur Erkennung von kardialem Risiko/ belastungsinduzierter Ischämie die Myokardszintigraphie zum Einsatz. ERGEBNISSE: Bei 28 Patienten (56 %) fanden sich pathologische Befunde in der Myokardszintigraphie. Davon zeigten 14 Patienten Zeichen einer sogenannten Small Vessel Disease (Kardiales Syndrom X). Darüber hinaus fanden sich weitere Begleiterkrankungen wie Adipositas, arterielle Hypertonie, Nikotinkonsum, Alkoholkonsum und erhöhte CRP-Werte. Die Häufigkeiten entsprachen im Wesentlichen den aktuellen Daten aus der Literatur. Wir konnten keinen signifikanten Zusammenhang von Schwere der Psoriasis oder der angegebenen Komorbiditäten mit einem pathologischen Befund in der Myokardszintigraphie feststellen. SCHLUSSFOLGERUNGEN: Mit der Myokardszintigraphie scheint ein sehr empfindliches, nicht invasives Früherkennungsverfahren zur Detektion kardialer Komorbidität bei Psoriasis-Patienten zur Verfügung zu stehen. Weitere größere Arbeiten mit Kontrollkollektiven und Kotrollmethoden, wie beispielsweise der Koronarangiographie, sind zur Überprüfung der Wertigkeit nötig.

Myocardial scintigraphy - a method for detecting cardiac comorbidity in psoriasis patients?

BACKGROUND: In recent years, cardiac comorbidities in psoriasis patients have increasingly moved into the focus of clinical research. The objective of the present study was to evaluate myocardial scintigraphy as a screening method in patients with psoriasis. PATIENTS AND METHODS: Assessment of various comorbidities in 50 psoriasis patients without clinical symptoms of cardiac disease. Myocardial scintigraphy was employed to detect cardiac risk/exercise-induced ischemia. RESULTS: Twenty-eight patients (56 %) had pathological findings on myocardial scintigraphy. Fourteen individuals showed evidence of small-vessel disease (cardiac syndrome X). Other comorbidities included obesity, arterial hypertension, nicotine and alcohol abuse, as well as elevated CRP levels. Frequencies largely corresponded to those reported in the recent literature. There was no significant correlation between the severity of psoriasis or any comorbidities and pathological findings on myocardial scintigraphy. CONCLUSIONS: Myocardial scintigraphy seems to be a very sensitive, noninvasive method for the early detection of cardiac comorbidities in psoriasis patients. However, determining its true diagnostic value will require larger studies with control subjects and control methods such as coronary angiography.

Spatially valid data of atmospheric deposition of heavy metals and nitrogen derived by moss surveys for pollution risk assessments of ecosystems.

For analysing element input into ecosystems and associated risks due to atmospheric deposition, element concentrations in moss provide complementary and time-integrated data at high spatial resolution every 5 years since 1990. The paper reviews (1) minimum sample sizes needed for reliable, statistical estimation of mean values at four different spatial scales (European and national level as well as landscape-specific level covering Europe and single countries); (2) trends of heavy metal (HM) and nitrogen (N) concentrations in moss in Europe (1990-2010); (3) correlations between concentrations of HM in moss and soil specimens collected across Norway (1990-2010); and (4) canopy drip-induced site-specific variation of N concentration in moss sampled in seven European countries (1990-2013). While the minimum sample sizes on the European and national level were achieved without exception, for some ecological land classes and elements, the coverage with sampling sites should be improved. The decline in emission and subsequent atmospheric deposition of HM across Europe has resulted in decreasing HM concentrations in moss between 1990 and 2010. In contrast, hardly any changes were observed for N in moss between 2005, when N was included into the survey for the first time, and 2010. In Norway, both, the moss and the soil survey data sets, were correlated, indicating a decrease of HM concentrations in moss and soil. At the site level, the average N deposition inside of forests was almost three times higher than the average N deposition outside of forests.