Occurrence of tardigrades and morphometric and chemical conditions in rock pools by the Baltic Sea.

Rock pools are eroded depressions in bedrock providing temporary aquatic habitats with varying morphometric and chemical conditions. Tardigrades have adapted to many habitats with varying and extreme abiotic conditions, including desiccation, but their occurrence in rock pools have rarely been investigated. This study investigated the occurrence of tardigrades and the morphometric and chemical conditions in rock pools by the Baltic Sea in southeast Sweden. Samples of benthic material were collected from rock pools at three sites near the town Karlshamn together with measurements of pool size, pH, temperature, salinity, and dissolved oxygen of the water. Tardigrades occurred in about one fifth of the rock pools and included five eutardigrade genera. Also rotifers and nematodes were observed in the samples. The morphometric and chemical variables varied both within and among the three sites but with few differences between rock pools with or without tardigrades. However, rock pools with tardigrades tended to be overall shallower than pools without tardigrades, indicating that more desiccating-prone rock pools may be more favourable habitats for tardigrades. The study shows that tardigrades are part of the micro-invertebrate fauna in rock pools and this habitat deserves more investigations into the occurrence of this animal group.

On the edge of decision-making in trauma care: A focus group study on radiographers' experiences of interprofessional collaboration.

INTRODUCTION: The temporary trauma teams in trauma alerts consist of a diverse group of unique professionals requiring interprofessional collaboration and coordination to achieve efficient, high-quality care. The uncertain situation and complex care environment impose high demands on team dynamics such as individual attitudes and team behaviours. Within interprofessional teams, interaction and coordination reflect the collective success of collaboration and the achievement of goals. Interactions with radiographers have increased in trauma teams given computed tomography's prominent role in providing crucial knowledge for decision-making in trauma care. This study aimed to explore radiographers' experiences of interprofessional collaboration during trauma alerts. METHOD: The study was designed with focus group methodology, including 17 radiographers participating in five focus groups, analysed with an inductive focus group analysis. RESULTS: An overarching theme, "On the edge of decision-making", emerged along with three sub-themes: "Feeling included requires acknowledgement", "Exclusion precludes shared knowledge", and "Experience and mutual awareness facilitate team interaction". CONCLUSIONS: Interprofessional collaboration from the radiographer's perspective within trauma teams requires a sense of inclusion and the ability to interact with the team. Exclusion from vital decision-making obstructs radiographers' comprehension of situations and thereby the interdependence in interprofessional collaboration. IMPLICATIONS FOR PRACTICE: Common platforms are needed for knowledge sharing and team practices, including radiographers' areas of responsibility and relational coordination to foster interprofessional relationships. Through these means interdependence through awareness and shared knowledge can be facilitated on trauma teams.

An experimental study on tolerance to hypoxia in tardigrades.

Introduction: Tardigrades are small aquatic invertebrates with well documented tolerance to several environmental stresses, including desiccation, low temperature, and radiation, and an ability to survive long periods in a cryptobiotic state under arrested metabolism. Many tardigrade populations live in habitats where temporary exposure to hypoxia is expected, e.g., benthic layers or substrates that regularly undergo desiccation, but tolerance to hypoxia has so far not been thoroughly investigated in tardigrades. Method: We studied the response to exposure for hypoxia (<1 ppm) during 1-24 h in two tardigrade species, Richtersius cf. coronifer and Hypsibius exemplaris. The animals were exposed to hypoxia in their hydrated active state. Results: Survival was high in both species after the shortest exposures to hypoxia but tended to decline with longer exposures, with almost complete failure to recover after 24 h in hypoxia. R. cf. coronifer tended to be more tolerant than H. exemplaris. When oxygen level was gradually reduced from 8 to 1 ppm, behavioral responses in terms of irregular body movements were first observed at 3-4 ppm. Discussion: The study shows that both limno-terrestrial and freshwater tardigrades are able to recover after exposure to severe hypoxia, but only exposure for relatively short periods of time. It also indicates that tardigrade species have different sensitivity and response patterns to exposure to hypoxia. These results will hopefully encourage more studies on how tardigrades are affected by and respond to hypoxic conditions.

Metrics for environmental compensation: A comparative analysis of Swedish municipalities.

Environmental compensation (EC) aims at addressing environmental losses due to development projects and involves a need to compare development losses with compensation gains using relevant metrics. A conceptual procedure for computing no net loss is formulated and used as a point of departure for a comparative analysis of metrics used by five Swedish municipalities as a part of their EC implementation in the spatial planning context of detailed development plans. While Swedish law does not require EC in this context, these municipalities have still decided to introduce EC requirements for development projects that occur on municipality-owned land and to promote voluntary EC among private actors in development projects on private land. There is substantial variation across the municipalities studied with respect to both metrics and attributes subject to measurement, but there are also similarities: The attributes considered when assessing the need for EC in conjunction with development are not only about nature per se, but also about recreational opportunities and other types ecosystem services; semi-quantitative metrics such as scores are common while quantitative or monetary metrics are rare; and metrics are rarely applied to assess compensatory gains, focusing instead on losses from development. Streamlining across municipalities might be warranted for increasing predictability and transparency for developers and citizens, but it also introduces considerable challenges such as a need for developing consistent guidelines for semi-quantitative metrics, and to handle substitutability issues if metrics are not only applied on individual attributes but also on groups of attributes. The broad scope of attributes used by the municipalities is in line with an international tendency to broaden EC to include not only biodiversity aspects but also ecosystem services. Moreover, the EC systems applied by the municipalities are of particular importance for highlighting the crucial role of environmental management for maintaining and enhancing biodiversity and ecosystem services not only in areas having formal protection status but also in the everyday landscape. The municipalities' experience and strengths and weaknesses associated with their EC systems are therefore relevant also in an international perspective.

Tardigrades of Kristianstads Vattenrike Biosphere Reserve with description of four new species from Sweden.

Kristianstads Vattenrike Biosphere Reserve [KVBR] is a UNESCO designated area of Sweden possessing high biological value. Although several studies on tardigrades inhabiting Sweden have been performed, the KVBR area has been neglected. The current study investigates the tardigrade fauna of five areas of the biosphere reserve and includes 34 samples of different substrates analysed quantitatively and qualitatively. In total, 33 species of tardigrades were found in the samples, including 22 new records for the Skåne region, 15 new records for Sweden, and four species new to science. Mesobiotus emiliae sp. nov., Xerobiotus gretae sp. nov., Itaquascon magnussoni sp. nov., and Thulinius gustavi sp. nov. were described with an integrative approach (when possible) using morphological characters (light, electron scanning, and confocal laser scanning microscopies) and molecular markers (ITS2, 18S, 28S, cox1). A new protocol to increase morphological data was developed recovering mounted specimens within old slides for SEM analysis. Emended diagnoses for the genus Itaquascon and the transfer of Platicrista itaquasconoide to the genus Meplitumen are proposed. This study enriches the knowledge of the tardigrade biodiversity both within the KVBR and in Sweden and contributes to the rapidly increasing number of tardigrade species reported worldwide. The 33 species identified in the KVBR area represents 28% of all water bear species found in Sweden so far. The restricted study areas and limited number of samples collected suggests that the KVBR is very rich of tardigrades.

Cell Biology of the Tardigrades: Current Knowledge and Perspectives.

The invertebrate phylum Tardigrada has received much attention for containing species adapted to the most challenging environmental conditions where an ability to survive complete desiccation or freezing in a cryptobiotic state is necessary for persistence. Although research on tardigrades has a long history, the last decade has seen a dramatic increase in molecular biological ("omics") studies, most of them with the aim to reveal the biochemical mechanisms behind desiccation tolerance of tardigrades. Several other aspects of tardigrade cell biology have been studied, and we review some of them, including karyology, embryology, the role of storage cells, and the question of whether tardigrades are eutelic animals. We also review some of the theories about how anhydrobiotic organisms are able to maintain cell integrity under dry conditions, and our current knowledge on the role of vitrification and DNA protection and repair. Many aspects of tardigrade stress tolerance have relevance for human medicine, and the first transfers of tardigrade stress genes to human cells have now appeared. We expect this field to develop rapidly in the coming years, as more genomic information becomes available. However, many basic cell biological aspects remain to be investigated, such as immunology, cell cycle kinetics, cell metabolism, and culturing of tardigrade cells. Such development will be necessary to allow tardigrades to move from a nonmodel organism position to a true model organism with interesting associations with the current models C. elegans and D. melanogaster.

Radiation Tolerance in Tardigrades: Current Knowledge and Potential Applications in Medicine.

Tardigrades represent a phylum of very small aquatic animals in which many species have evolved adaptations to survive under extreme environmental conditions, such as desiccation and freezing. Studies on several species have documented that tardigrades also belong to the most radiation-tolerant animals on Earth. This paper gives an overview of our current knowledge on radiation tolerance of tardigrades, with respect to dose-responses, developmental stages, and different radiation sources. The molecular mechanisms behind radiation tolerance in tardigrades are still largely unknown, but omics studies suggest that both mechanisms related to the avoidance of DNA damage and mechanisms of DNA repair are involved. The potential of tardigrades to provide knowledge of importance for medical sciences has long been recognized, but it is not until recently that more apparent evidence of such potential has appeared. Recent studies show that stress-related tardigrade genes may be transfected to human cells and provide increased tolerance to osmotic stress and ionizing radiation. With the recent sequencing of the tardigrade genome, more studies applying tardigrade omics to relevant aspects of human medicine are expected. In particular, the cancer research field has potential to learn from studies on tardigrades about molecular mechanisms evolved to maintain genome integrity.

Phobos LIFE (Living Interplanetary Flight Experiment).

The Planetary Society's Phobos Living Interplanetary Flight Experiment (Phobos LIFE) flew in the sample return capsule of the Russian Federal Space Agency's Phobos Grunt mission and was to have been a test of one aspect of the hypothesis that life can move between nearby planets within ejected rocks. Although the Phobos Grunt mission failed, we present here the scientific and engineering design and motivation of the Phobos LIFE experiment to assist with the scientific and engineering design of similar future experiments. Phobos LIFE flew selected organisms in a simulated meteoroid. The 34-month voyage would have been the first such test to occur in the high-radiation environment outside the protection of Earth's magnetosphere for more than a few days. The patented Phobos LIFE "biomodule" is an 88 g cylinder consisting of a titanium outer shell, several types of redundant seals, and 31 individual Delrin sample containers. Phobos LIFE contained 10 different organisms, representing all three domains of life, and one soil sample. The organisms are all very well characterized, most with sequenced genomes. Most are extremophiles, and most have flown in low Earth orbit. Upon return from space, the health and characteristics of organisms were to have been compared with controls that remained on Earth and have not yet been opened.

Drivers of changing urban flood risk: A framework for action.

This study focuses on drivers for changing urban flood risk. We suggest a framework for guiding climate change adaptation action concerning flood risk and manageability in cities. The identified key drivers of changing flood hazard and vulnerability are used to provide an overview of each driver's impact on flood risk and manageability at the city level. We find that identified drivers for urban flood risk can be grouped in three different priority areas with different time horizon. The first group has high impact but is manageable at city level. Typical drivers in this group are related to the physical environment such as decreasing permeability and unresponsive engineering. The second group of drivers is represented by public awareness and individual willingness to participate and urbanization and urban sprawl. These drivers may be important and are manageable for the cities and they involve both short-term and long-term measures. The third group of drivers is related to policy and long-term changes. This group is represented by economic growth and increasing values at risk, climate change, and increasing complexity of society. They have all high impact but low manageability. Managing these drivers needs to be done in a longer time perspective, e.g., by developing long-term policies and exchange of ideas.

A comparative ultrastructure study of storage cells in the eutardigrade Richtersius coronifer in the hydrated state and after desiccation and heating stress.

Tardigrades represent an invertebrate phylum with no circulatory or respiratory system. Their body cavity is filled with free storage cells of the coelomocyte-type, which are responsible for important physiological functions. We report a study comparing the ultrastructure of storage cells in anhydrobiotic and hydrated specimens of the eutardigrade Richtersius coronifer. We also analysed the effect of temperature stress on storage cell structure. Firstly, we verified two types of ultrastructurally different storage cells, which differ in cellular organelle complexity, amount and content of reserve material and connection to oogenetic stage. Type I cells were found to differ ultrastructurally depending on the oogenetic stage of the animal. The main function of these cells is energy storage. Storage cells of Type I were also observed in the single male that was found among the analysed specimens. The second cell type, Type II, found only in females, represents young undifferentiated cells, possibly stem cells. The two types of cells also differ with respect to the presence of nucleolar vacuoles, which are related to oogenetic stages and to changes in nucleolic activity during oogenesis. Secondly, this study revealed that storage cells are not ultrastructurally affected by six months of desiccation or by heating following this desiccation period. However, heating of the desiccated animals (tuns) tended to reduce animal survival, indicating that long-term desiccation makes these animals more vulnerable to heat stress. We confirmed the degradative pathways during the rehydration process after desiccation and heat stress. Our study is the first to document two ultrastructurally different types of storage cells in tardigrades and reveals new perspectives for further studies of tardigrade storage cells.

On the road to 'research municipalities': analysing transdisciplinarity in municipal ecosystem services and adaptation planning.

Transdisciplinary research and collaboration is widely acknowledged as a critical success factor for solution-oriented approaches that can tackle complex sustainability challenges, such as biodiversity loss, pollution, and climate-related hazards. In this context, city governments' engagement in transdisciplinarity is generally seen as a key condition for societal transformation towards sustainability. However, empirical evidence is rare. This paper presents a self-assessment of a joint research project on ecosystem services and climate adaptation planning (ECOSIMP) undertaken by four universities and seven Swedish municipalities. We apply a set of design principles and guiding questions for transdisciplinary sustainability projects and, on this basis, identify key aspects for supporting university-municipality collaboration. We show that: (1) selecting the number and type of project stakeholders requires more explicit consideration of the purpose of societal actors' participation; (2) concrete, interim benefits for participating practitioners and organisations need to be continuously discussed; (3) promoting the 'inter', i.e., interdisciplinary and inter-city learning, can support transdisciplinarity and, ultimately, urban sustainability and long-term change. In this context, we found that design principles for transdisciplinarity have the potential to (4) mitigate project shortcomings, even when transdisciplinarity is not an explicit aim, and (5) address differences and allow new voices to be heard. We propose additional guiding questions to address shortcomings and inspire reflexivity in transdisciplinary projects.

Neonatal intestinal colonization with extended-spectrum ß-lactamase-producing Enterobacteriaceae-a 5-year follow-up study.

OBJECTIVES: To analyse Klebsiella pneumoniae (KP) isolates from an outbreak of extended-spectrum ß-lactamase (ESBL)-producing KP and Escherichia coli (EC) among infants admitted to neonatal intensive care units and to determine the duration of the intestinal colonization. METHODS: We performed a prospective cohort study of intestinal ESBL-KP/ESBL-EC colonized neonates after a 5-month outbreak in two neonatal intensive care units. Whole genome sequencing, multilocus sequence typing, core genome multilocus sequence typing, pulsed-field electrophoresis and PCR for blaCTX-M were performed on the first isolates. Stool cultures were performed every second month after discharge until 2 years after discharge and at 5 years of age. The last positive samples were analysed with pulsed-field gel electrophoresis and PCR for blaCTX-M. The intestinal relative dominance of ESBL-producing Enterobacteriaceae was determined. RESULTS: Thirteen of 17 patients colonized with ESBL-KP/ESBL-EC survived. Isolates from 16 of 17 patients were available for analysis and featured the same strain type of ESBL-KP: sequence type 101. The strain had capsule type K29 and harboured blaCTX-M-15. The virulence genes irp1, irp2, iutA, kfu and mrk were detected in all isolates. The median length of colonization was 12.5 months (range, 5-68 months). After 2 years, two of 13 patients were carriers of ESBL-KP and one of 13 of ESBL-EC. At 5 years of age, one neonate was colonized with ESBL-EC. No infant experienced an ESBL-KP/EC-infection during follow-up. CONCLUSIONS: Two years after discharge, almost one fourth of the study participants were ESBL/KP-EC carriers. ESBL-KP sequence type 101 persisted in two of 13 children for 23 to 26 months. One patient was colonized with ESBL-EC at age 5 years.

Late introduction of fish and eggs is associated with increased risk of allergy development - results from the FARMFLORA birth cohort.

The prevalence of allergy is markedly low in children growing up on farms. An increasing number of studies indicate that the timing of food introduction may affect allergy development. We aimed to investigate if protection against allergy in farm environments may be mediated through differences in food-introduction practices between farm and non-farm families, using an explorative approach. Twenty-eight farm and 37 non-farm children were included in the FARMFLORA birth cohort. Practices of breastfeeding and introduction of formulas and complementary foods were collected by questionnaires at 6, 12, and 18 months of age. Allergy was diagnosed by pediatricians at 3 years of age. The only difference in food-introduction practices observed between farm and non-farm children was an earlier introduction of nuts in farmers (median month: 11 [IQR: 8-6] in farmers, 15 [12-19] in non-farmers). One farm child (4%) and 10 non-farm children (27%) were allergic at 3 years of age. Lower risk of allergy development was associated with early exclusive breastfeeding (continuous variable; OR = 0.59, 95% CI: 0.39-0.89), but also having received eggs (OR = 0.08, 95% CI: 0.13-0.54) and fish (logistic regression not applicable, P = 0.01 in likelihood ratio testing [χ2]) at 10 months of age or earlier compared to later. Our results were not affected by reverse causation, as judged by a questionnaire sent to the families in retrospect. Timing of introduction of complementary foods is unlikely to contribute to the lower risk of allergy among farm children. Although early exclusive breastfeeding was associated with a lower rate of allergy development, postponed introduction of complementary foods might increase the risk of developing allergy. Owing to the limited sample size, our results are only indicative, but support prior findings.

Fostering incidental experiences of nature through green infrastructure planning.

Concern for a diminished human experience of nature and subsequent decreased human well-being is addressed via a consideration of green infrastructure's potential to facilitate unplanned or incidental nature experience. Incidental nature experience is conceptualized and illustrated in order to consider this seldom addressed aspect of human interaction with nature in green infrastructure planning. Special attention has been paid to the ability of incidental nature experience to redirect attention from a primary activity toward an unplanned focus (in this case, nature phenomena). The value of such experience for human well-being is considered. The role of green infrastructure to provide the opportunity for incidental nature experience may serve as a nudge or guide toward meaningful interaction. These ideas are explored using examples of green infrastructure design in two Nordic municipalities: Kristianstad, Sweden, and Copenhagen, Denmark. The outcome of the case study analysis coupled with the review of literature is a set of sample recommendations for how green infrastructure can be designed to support a range of incidental nature experiences with the potential to support human well-being.

IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP.

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses.

Tolerance to X-rays and Heavy Ions (Fe, He) in the Tardigrade Richtersius coronifer and the Bdelloid Rotifer Mniobia russeola.

The aim of this study was to analyze tolerance to heavy ions in desiccated animals of the eutardigrade Richtersius coronifer and the bdelloid rotifer Mniobia russeola within the STARLIFE project. Both species were exposed to iron (Fe) and helium (He) ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) in Chiba, Japan, and to X-rays at the German Aerospace Center (DLR) in Cologne, Germany. Results show no effect of Fe and He on viability up to 7 days post-rehydration in both R. coronifer and M. russeola, while X-rays tended to reduce viability in R. coronifer at the highest doses. Mean egg production rate tended to decline with higher doses in R. coronifer for all radiation types, but the pattern was not statistically confirmed. In M. russeola, there was no such tendency for a dose response in egg production rate. These results confirm the previously reported high tolerance to high linear energy transfer (LET) radiation in tardigrades and show for the first time that bdelloid rotifers are also very tolerant to high-LET radiation. These animal phyla represent the most desiccation- and radiation-tolerant animals on Earth and provide excellent eukaryotic models for astrobiological research. Key Words: Tardigrada-Rotifera-Radiation tolerance-Heavy ions-X-rays. Astrobiology 17, 163-167.

The structure of the desiccated Richtersius coronifer (Richters, 1903).

Tun formation is an essential morphological adaptation for entering the anhydrobiotic state in tardigrades, but its internal structure has rarely been investigated. We present the structure and ultrastructure of organs and cells in desiccated Richtersius coronifer by transmission and scanning electron microscopy, confocal microscopy, and histochemical methods. A 3D reconstruction of the body organization of the tun stage is also presented. The tun formation during anhydrobiosis of tardigrades is a process of anterior-posterior body contraction, which relocates some organs such as the pharyngeal bulb. The cuticle is composed of epicuticle, intracuticle and procuticle; flocculent coat; and trilaminate layer. Moulting does not seem to restrict the tun formation, as evidenced from tardigrade tuns that were in the process of moulting. The storage cells of desiccated specimens filled up the free inner space and surrounded internal organs, such as the ovary and digestive system, which were contracted. All cells (epidermal cells, storage cells, ovary cells, cells of the digestive system) underwent shrinkage, and their cytoplasm was electron dense. Lipids and polysaccharides dominated among reserve material of storage cells, while the amount of protein was small. The basic morphology of specific cell types and organelles did not differ between active and anhydrobiotic R. coronifer.

Tolerance to Gamma Radiation in the Marine Heterotardigrade, Echiniscoides sigismundi.

Tardigrades belong to the most radiation tolerant animals on Earth, as documented by a number of studies using both low-LET and high-LET ionizing radiation. Previous studies have focused on semi-terrestrial species, which are also very tolerant to desiccation. The predominant view on the reason for the high radiation tolerance among these semi-terrestrial species is that it relies on molecular mechanisms that evolved as adaptations for surviving dehydration. In this study we report the first study on radiation tolerance in a marine tardigrade, Echiniscoides sigismundi. Adult specimens in the hydrated active state were exposed to doses of gamma radiation from 100 to 5000 Gy. The results showed little effect of radiation at 100 and 500 Gy but a clear decline in activity at 1000 Gy and higher. The highest dose survived was 4000 Gy, at which ca. 8% of the tardigrades were active 7 days after irradiation. LD50 in the first 7 days after irradiation was in the range of 1100-1600 Gy. Compared to previous studies on radiation tolerance in semi-terrestrial and limnic tardigrades, Echiniscoides sigismundi seems to have a lower tolerance. However, the species still fits into the category of tardigrades that have high tolerance to both desiccation and radiation, supporting the hypothesis that radiation tolerance is a by-product of adaptive mechanisms to survive desiccation. More studies on radiation tolerance in tardigrade species adapted to permanently wet conditions, both marine and freshwater, are needed to obtain a more comprehensive picture of the patterns of radiation tolerance.

Experimentally Induced Repeated Anhydrobiosis in the Eutardigrade Richtersius coronifer.

Tardigrades represent one of the main animal groups with anhydrobiotic capacity at any stage of their life cycle. The ability of tardigrades to survive repeated cycles of anhydrobiosis has rarely been studied but is of interest to understand the factors constraining anhydrobiotic survival. The main objective of this study was to investigate the patterns of survival of the eutardigrade Richtersius coronifer under repeated cycles of desiccation, and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed potential change in body size, gut content and frequency of mitotic storage cells. Specimens were kept under non-cultured conditions and desiccated under controlled relative humidity. After each desiccation cycle 10 specimens were selected for analysis of morphometric characteristics and mitosis. The study demonstrates that tardigrades may survive up to 6 repeated desiccations, with declining survival rates with increased number of desiccations. We found a significantly higher proportion of animals that were unable to contract properly into a tun stage during the desiccation process at the 5th and 6th desiccations. Also total number of storage cells declined at the 5th and 6th desiccations, while no effect on storage cell size was observed. The frequency of mitotic storage cells tended to decline with higher number of desiccation cycles. Our study shows that the number of consecutive cycles of anhydrobiosis that R. coronifer may undergo is limited, with increased inability for tun formation and energetic constraints as possible causal factors.

Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.