The top-down approach to measurement uncertainty: which formula should we use in laboratory medicine?

Introduction: By quantifying the measurement uncertainty (MU), both the laboratory and the physician can have an objective estimate of the results’ quality. There is significant flexibility on how to determine the MU in laboratory medicine and different approaches have been proposed by Nordtest, Eurolab and Cofrac to obtain the data and apply them in formulas. The purpose of this study is to compare three different top-down approaches for the estimation of the MU and to suggest which of these approaches could be the most suitable choice for routine use in clinical laboratories. Materials and methods: Imprecision and bias of the methods were considered as components of the MU. The bias was obtained from certified reference calibrators (CRC), proficiency tests (PT), and inter-laboratory internal quality control scheme (IQCS) programs. The bias uncertainty, the combined and the expanded uncertainty were estimated using the Nordtest, Eurolab and Cofrac approaches. Results: Using different approaches, the expanded uncertainty estimates ranged from 18.9-40.4%, 18.2-22.8%, 9.3-20.9%, and 7.1-18.6% for cancer antigen (CA) 19-9, testosterone, alkaline phosphatase (ALP), and creatinine, respectively. Permissible values for MU and total error ranged from 16.0-46.1%, 13.1-21.6%, 10.7-26.2%, and 7.5-17.3%, respectively. Conclusion: The bias was highest using PT, followed by CRC and IQCS data, which were similar. The Cofrac approach showed the highest uncertainties, followed by Eurolab and Nordtest. However, the Eurolab approach requires additional measurements to obtain uncertainty data. In summary, the Nordtest approach using IQCS data was therefore found to be the most practical formula

Phylogeographic study of brown trout from Serbia, based on mitochondrial DNA control region analysis

In order to illuminate the phylogeography of brown trout (Salmo trutta) populations in the Balkan state of Serbia, the 561 bp 5'-end of mtDNA control region of 101 individuals originating from upland tributaries of the Danubian, Aegean and Adriatic drainages were sequenced and compared to corresponding brown trout sequences obtained in previous studies. Among 15 haplotypes found, 14 were considered native, representing the Danubian and Adriatic lineages of the brown trout, while one haplotype (ATcs1), found only in two individuals originating from two stocked rivers, corresponded to the Atlantic lineage and was considered introduced. Native haplotypes exhibited a strong geographic pattern of distribution: the Danubian haplotypes were strictly confined to the Danubian drainage, while the Adriatic haplotypes dominated in the Aegean and Adriatic drainages; most of the total molecular variance (69%) was attributed to differences among the drainages. Phylogenetic reconstruction, supplemented with seven haplotypes newly described in this study, suggested a sister position of the Atlantic-Danubian and Adriatic-Mediterranean-marmoratus ("southern") phylogenetic group, and pointed to the existence of a distinct clade, detected within the "southern" group. The data obtained confirmed our expectation of the existence of high genetic diversity in Balkan trout populations, and we recommend more widespread surveys covering trout stocks from the region

Analiza genetičke strukture mladice (Hucho hucho) u srbiji na osnovu mitohondrijske i jedarne DNK

The purpose of this study was to assess the genetic diversity of Serbian huchen and provide guidelines for conservation and management. Fourteen huchen individuals were collected from six localities. For all individuals the mitochondrial DNA control region and the NADH1 gene were sequenced, and two microsatellite loci were genotyped. Sequencing of two mtDNA loci showed the presence of the same haplotypes (Hh_CR_1 and Hh_ND1_1) in all investigated specimens and the complete absence of genetic variability at inter- and intra-populational level. However, analyzed individuals from Serbia have the largest number of control region repeats (average 8.7) compared to all other huchen populations from the Danube basin. Microsatellite data show that the most of Serbian huchens were placed in the “eastern” cluster except one individual from the Drina River which clustered with “western” samples, most likely a consequence of stocking. In spite of only two loci used, four unique alleles found in the studied Serbian and Drina basin population indicate that the Danube basin may possess more region specific alleles than previously thought, which could be of great importance for efficient species management in the future.Cilj ovoga rada je bio da se izvrši procena genetičkog diverziteta mladice u Srbiji, i da se na osnovu dobijenih rezultata daju preporuke za buduće očuvanje i upravljanje fondom mladice. Za ovu svrhu sakupljeno je 14 jedinki sa šest lokaliteta. Za sve sakupljene jedinke sekvencirana je kontrolna regija, NADH1 gen mitohondijske DNK i genotipizirana su dva mikrosatelitska lokusa. Sekvenciranjem dva mtDNK lokusa utvrđeno je prisustvo istih haplotipova (Hh_CR_1 i Hh_ND1_1) kod svih ispitanih jedinki, odnosno, potpuno odsustvo genetičke varijabilnosti na inter- i intra-populacionom nivou. Međutim, analizirane jedinke iz Srbije karakterišu se najvećim brojem ponovaka kontrolne regije (prosečno 8,7), u poređenju sa svim ostalim populacijama mladice iz dunavskog sliva. Analizom mikrosatelitskih podataka, utvrđeno je da se gotovo sve jedinke iz Srbije svrstavaju u “istočnu” grupu, izuzev jedne jedinke iz Drine, koja je svrstana sa “zapadnim” uzorcima, što je najverovatnije posledica poribljavanja. Kod analiziranih jedinki sa teritorije Srbije i iz celokupnog sliva Drine pronađena su četiri jedinstvena alela, uprkos analizi samo dva mikrosatelitska lokusa. Dobijeni rezultati ukazuju da dunavski sliv može posedovati više regionalno specifičnih alela nego što se ranije smatralo, što bi moglo biti od velikog značaja za uspešno upravljanje fondom mladice u budućnosti

Cryopreservation of sperm of the adriatic grayling (Thymallus thymallus) and the marble trout (Salmo marmoratus) from the Soča river in Slovenia

A cryopreservation protocol was tested on the sperm of genetically distinct Adriatic grayling (Thymallus thymallus) and the Natura2000-listed marble trout (Salmo marmoratus) from the Soča River basin in Slovenia. Sperm was frozen in an extender composed of 200 mM glucose, 40 mM KCl, 30 mM Tris (pH 8.0) and 10% methanol as a cryoprotectant in the vapour of liquid nitrogen. Sperm dilution ratios of 1:1, 1:4 and 1:9 were tested. The cooling rate was 57 ± 1°Cmin-1 in all cases. In the grayling, the highest ratio of eyed eggs (74 ± 4 % vs. 69 ± 6 % in the control) and the highest hatch percentage (63 ± 6 % vs. 56 ± 10 % in the control) was observed with a dilution ratio of 1:1. In the marble trout, the highest percentage of eyed eggs (84 ± 4 % vs. 88 ± 3 in the control) and the highest hatch rate (70 ± 3 % vs. 76 ± 2 % in the control) was again found using the 1:1 dilution ratio of the sperm. In both species, individual sperm samples had a significant effect on the results. Sperm-to-egg ratios ranging from 2.5 to 5.7 × 104 spermatozoa per egg yielded satisfactory hatch rates (67 ± 7 % to 73 ± 7 %) in the grayling

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus

Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna

Molecular data as a possible tool for tracing groundwater flow in karst environment

Karst areas have some of the most complex aquifers in the world. Because of extreme groundwater oscillations, different hydrological connections are active depending on the season. Therefore, the complex water network remains poorly investigated, despite many geological, hydrological and geomorphological studies performed. The methodology of water tracing was developed especially to determine water interconnections in karst. In additionto tracing with salt, dye or temperature, the use of rich endemic fauna, their dispersion and population structure is lately considered. In this paper, we used population structure of endemic fish species Delminichthys adspersus to confirm hydrological data as well as suggest new underground water connections in part of central Dinaric karst. Moreover, we propose a holistic approach of karst area studies, combining hydrology with biology to develop a better strategy for the protection of vulnerable karst ecosystems

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus

Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna

Regional structure despite limited mtDNA sequence diversity found in the endangered Huchen, Hucho hucho (Linnaeus, 1758)

We evaluate the hypothesis of no geographic structure in the Huchen (Hucho hucho), a large, predatory salmonid endemic to the Danube basin. Forty-seven individuals sampled from throughout the Hauchen\u27s native range were genetically characterized. Extremely limited sequence diversity across 1,800 bases of mtDNA (the complete control region and partial NADH-1 subunit) evidenced by four closely related mtDNA haplotypes was found. Nonetheless, the geographic distribution of mtDNA repeats (5-10, 82-bp long copies per individual) as well as allelic diversity across two microsatellite loci indicated large-scale geographic structure between the north-western (Austria and Slovenia) distribution area and eastern Slovakia nad Ukraine) or southern (Bosnia-Herzegovina and Montenegro) sample sites. An extremly slow rate of substitution for the H. hucho mtDNA is considered along with the alternative hypotheses to explain the limited mtDNA diversity. Considering the regional genetic structure implied by our data, we advocate restrictions on the transport of brood fish of yearlings across the range of the species distribution and sale of Huchen across international boundaries. Future genetic analysis to support local conservation and monitoring efforts must focus on developing a high-resolution screen that may be applied to identify hatchery versus naturally reproduction individuals in the wild