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Influenza A virus (IAV) is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu in humans. Its genome comprises eight RNA segments that facilitate reassortment, resulting in a great variety of IAV strains. To study these processes, the genetic code of each segment should be unraveled. Fortunately, new third-generation sequencing approaches allow for cost-efficient sequencing of IAV segments. Sequencing success depends on various factors, including proper sample storage and processing. Hence, this work focused on the effect of storage of oral fluids and swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at −22 °C and later transferred to −80 °C. Other samples (n = 21) were immediately stored at −80 °C. A reverse transcription quantitative PCR (RT-qPCR) pre- and post-storage was conducted to assess IAV viral loads. Next, samples were subjected to two IAV long-read nanopore sequencing methods to evaluate success in this complex matrix. A significant storage-associated loss of swIAV loads was observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U (H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In conclusion, oral fluids can be used for long-read swIAV sequencing when considering appropriate storage and segment amplification protocols, which allows us to monitor swIAV in an animal-friendly and cost-efficient manner.

Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 healthy adult women, we investigated the microbiome preservation efficacy of AssayAssure® Genelock (as opposed to no preservative) under different temperature conditions. We varied temperature over 48 hours in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: -20C, ambient temperature, 4C, a freeze-thaw cycle, and a heat cycle. At 48 hours, all samples were stored at -80C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing AssayAssure® Genelock vs. no preservative, we did observe a higher concordance with AssayAssure samples shipped at colder temperatures (−20C and 4C) when compared to the samples shipped at -20C without preservative. Our results indicate that AssayAssure does not introduce a significant amount of microbial bias when used on a range of temperatures but is most effective at colder temperatures.ImportanceThe urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile: urinary tract microbes have been linked to health problems including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion respectively spent in the U.S. annually. Given the low biomass of urine, novelty of the field, and well-established replicability bias in microbiome studies, it is critical to study storage conditions on urine samples to minimize microbial biases. Efficient and reliable preservation methods permit home self-sample collection and shipping, increasing the accessibility of larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.

Catecholamine release increases in dogs with pheochromocytomas and in situations of stress. Although plasma catecholamines degrade rapidly, their metabolites, normetanephrine (NME) and metanephrine (ME), are stable in acidified urine. Our aim was to verify a human urine ELISA kit for the quantification of NME and ME in canine urine and to determine the effects on metabolite stability of sampling time (morning or midday) and day (ordinary or day spent in a clinic). We analyzed 179 urine samples from 17 healthy dogs. For NME, the mean intra-assay CV was 6.0% for all samples and 4.3% for the canine control; inter-assay CVs were 3.3, 3.8, and 12% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 90–101%. For ME, mean intra-assay CV was 6.5% for samples and 9.0% for the canine control; inter-assay CVs were 12.7, 7.2, and 22.5% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 85–89%. Dilution recovery was unsatisfactory for both metabolites. Based on our verification results, NME was selected for remaining analyses. We found no effect on NME concentrations of acidification or room temperature storage for up to 24 h. The NME:creatinine ratio was higher after the first of 3 clinic days compared to the same morning (111.2 ± 5.5 vs. 82.9 ± 5.3; p

Source web page: https://www.globalgeochemicalbaselines.eu/content/174/iugs-manual-of-standard-methods-for-establishing-the-global-geochemical-reference-network-/ SYNOPSIS “Everything in and on the Earth - mineral, animal and vegetable - is made from one, or generally some combination of, the natural chemical elements occurring in the rocks of the Earth’s crust and the surficial materials derived from them. Everything that is grown, or made, depends upon the availability of the appropriate elements. The existence, quality and survival of life depends upon the availability of elements in the correct proportions and combinations. Because natural processes and human activities are continuously modifying the chemical composition of our environment, it is important to determine the present abundance and spatial distribution of the elements across the Earth’s surface in a much more systematic manner than has been attempted hitherto” (Darnley et al., 1995, p.x). Although such a global database is urgently needed for multi-purpose use, the systematic attempt is still in its infancy because of the non-existence of a manual of comprehensive and standardised methods of sampling and other supporting procedures. The current ‘International Union of Geological Sciences Manual of Standard Methods for Establishing the Global Geochemical Reference Network’ fills this gap. The Manual follows the concept of 7356 Global Terrestrial Network grid cells of 160x160 km, covering the land surface of Earth, with five random sites within each grid cell for the collection of samples. This allows the establishment of the standardised Global Geochemical Reference Network with respect to rock, residual soil, humus, overbank sediment, stream water, stream sediment and floodplain sediment. Apart from the instructions for the collection of samples, the Manual covers sample preparation and storage, development of reference materials, geoanalytical methods, quality control procedures, geodetic and parametric levelling of existing geochemical data sets, data conditioning for the generation of time-independent geochemical data, management of data and map production, and finally project management. This Manual presents, for the first time a comprehensive overview of the standardised methods that should be employed across the land surface of the Earth to map the distribution of chemical elements in rock, soil, sediment and water. The methods described herein, apart from their use for Establishing the Global Geochemical Reference Network, can be used in other geochemical surveys at any mapping scale. The URL of the pdf file is: https://www.globalgeochemicalbaselines.eu/datafiles/file/IUGS-CGGB_2022_Manual_of_Standard_Methods_for_Establishing_the_GGRN.pdf IUGS APPROVAL FOR PUBLICATION The Executive Committee of the International Union of Geological Sciences at its 77th meeting in Paris (16-18 March 2022), unanimously approved the “International Union of Geological Sciences Manual of Standard Methods for Establishing the Global Geochemical Reference Network” to be published as a formal IUGS Publication for its 60th Anniversary celebration. The IUGS Executive Committee members John Ludden (President), Hassina Mouri (Vice President), Daekyo Cheong (Vice President), Stanley C. Finney (Secretary-General), Hiroshi Kitazato (Treasurer), Qiuming Cheng (Past President), Silvia Peppoloni (Councillor), Claudia Inés Mora (Councillor), Jennifer McKinley (Councillor), and Ludwig Stroink (Councillor) are thanked for approving the publication of this Manual. Supplementary material The supplementary material provided in different Chapters and Annexes is very important and should be consulted: Chapter 2 Supplementary material Chapter 2 Annexe A2.1 Supplementary material Chapter 3.2 Annexe A3.2.1 Supplementary material Chapter 7 Supplementary material Chapter 8 Supplementary material An innovation for this freely available publication is the provision of the original MicrosoftTM Word files, Original figures, and PowerPoint presentations with embedded figures: Chapter files: Word text; Original figures; PowerPoint presentations, CITATION: Demetriades, A., Johnson, C.C., Smith, D.B., Ladenberger, A., Adánez Sanjuan, P., Argyraki, A., Stouraiti, C., Caritat, P. de, Knights, K.V., Prieto Rincón, G. & Simubali, G.N. (Editors), 2022. International Union of Geological Sciences Manual of Standard Methods for Establishing the Global Geochemical Reference Network. IUGS Commission on Global Geochemical Baselines, Athens, Hellenic Republic, Special Publication, 2, 515 pp.; https://doi.org/10.5281/zenodo.7307696.

EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, −20 °C, and −80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing −80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at −80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

Latar belakang: Leptospirosis merupakan zoonosis penting di dunia, yang masih sering terjadi salah diagnosis. Deteksi laboratorium Leptospira menjadi tantangan karena bakterimea cukup singkat untuk dideteksi molekuler, namun antibodi juga muncul sangat lambat. Urine dapat menjadi sampel alternatif untuk deteksi PCR pada leptospirosis. Pengerjaan PCR membutuhkan DNA berkualitas dan andal, dan diperoleh dari metode ekstraksi DNA yang baik. Penelitian bertujuan untuk mengetahui metode ekstraksi DNA Leptospira terbaik untuk sampel urin, serta mengevaluasi pengaruh waktu penyimpanan dan suhu terhadap kestabilan DNA. Metode: Penelitian ini menggunakan tiga metode isolasi DNA yang berbeda; berbasis silika dengan spin kolom, kromatografi spin column menggunakan resin sebagai matriks pemisah, dan metode larutan dengan guanidine isothiocyanate. Hasil ekstraksi diperiksa konsentrasi dan kemurniannya. Gen SecY pada Leptospira dideteksi dengan PCR real-time. Pengaruh suhu dan lama penyimpanan DNA juga dilihat. Hasil: Hasil isolasi DNA menggunakan resin menunjukkan konsentrasi tertinggi (7,94 + 2,11 μg / mL) dan jumlah salinan amplifikasi DNA Leptospira tertinggi (50167,92 + 1,19). Suhu penyimpanan pada suhu 4°C, -20°C, dan -80°C dan umur simpan 91 hari tidak berpengaruh terhadap kualitas dan kuantitas DNA Leptospira hasil isolasi spike urin. Kesimpulan: Isolasi DNA menggunakan spin column chromatography dengan resin sebagai matriks separasi memiliki kualitas dan kuantitas terbaik berdasarkan kemurnian dan konsentrasi DNA serta jumlah gen SecY yang teramplifikasi. Kata kunci: Leptospira, Leptospirosis, ekstraksi DNA, sampel urin, penyimpanan sampel. Abstract Background: Leptospirosis is a worldwide zoonotic disease, which is still often misdiagnosed. Laboratory detection of Leptospira is challenging since the bacteraemia is quite short for molecular detection, however, the rise of the antibody is late to post the infection. Urine can be a potential alternative sample for PCR detection in leptospirosis. The PCR method requires a reliable DNA template, which is obtained from good DNA extracting methods. The study aimed to determine the best method of extraction Leptospira DNA from the urine sample, as well as evaluating the effect of time storage and temperature for its DNA stability. Methods: This study was utilizing three different DNA isolation methods; silica based with spin column, spin column chromatography using resin as separation matrix, and solution method with guanidine isothiocyanate. The yields were examined for its concentration and purity. Leptospira’s SecY gene was detected with realtime PCR. The influences of storage temperature and the life time of the DNA were also studied. Results: The yield of DNA isolation using resin showed the highest concentration (7.94+2.11 μg/mL) and highest Leptospira DNA amplification copy number (50167.92+1.19). Storage temperature at 4°C, -20°C, and -80°C and life time of 91 days did not have any effect on the quality and quatnity of Leptospira DNA isolated from spiked urine. Conclusions: DNA isolation using spin column chromatography with resin as separation matrix has the best quality and quantity based on the purity and concentration of DNA and the higher number of amplified SecY gene. Keywords: Leptospira, Leptospirosis, DNA extraction, urine sample, sample storage

In health surveys, especially in health examination surveys (HES), biological samples are commonly collected and often also stored for future use. This provides a possibility to extend the health surveys also to HBM if underlying criteria for the sample collection and supporting information is met. This document will provide a short overview of what is known about availability of stored samples in national HESs in Europe, what are the key criteria for samples when analysing the HBM4EU 1st priority chemicals, and what are the criteria for the use of stored biological samples from health studies for HBM analysis

Knjižnice so glavni vir informacij, katerih uporaba je namenjena hranjenju zapisov, knjig, znanstvenih člankov in ostalih stvari povezanih z informacijami. Posledica razvoja znanosti je kopičenje novih informacij, kar je povzročilo razvoj knjižnic. Sočasno z razvojem znanosti so se razvile tudi specificirane knjižnice, tako so se ob razvoju kemijske znanosti oblikovale tudi knjižnice kemijskih spojin. Vzorci in podatki v knjižnicah spojin so med drugim namenjeni hranjenju spojin, dostopu do informacij, nadaljnji sintezi spojin, raziskavam novih materialov ali odkrivanju novih medicinskih učinkovin. V sklopu Fakultete za Kemijo in Kemijsko Tehnologijo Univerze v Ljubljani, se je leta 2018 oblikovala Knjižnica spojin FKKT. Knjižnica ima tako fizično knjižnico, kot virtualno knjižnico, v slednji so zabeležene strukture spojin iz fizične knjižnice spojin. V virtualni knjižnici so zavedeni še izračunani opisniki (»deskriptorji«) spojin. Knjižnica spojin FKKT je vodena ročno in vsebuje spojine sintetizirane na Fakulteti za Kemijo in Kemijsko tehnologijo. Vzorci v knjižnici spojin FKKT so pretežno organske narave. S pomočjo NMR analize vzorcev sem določil, da je čistih okrog 85 % spojin v knjižnici, medtem ko so ostali razpadli ali imajo prisotne nečistoče. Z virtualnim rešetanjem struktur iz virtualne knjižnice spojin smo analizirali potencial vzorcev v knjižnici za uporabo v medicini. Kot biološko pomembno tarčo smo uporabili 3CLpro, proteazo pomembno pri replikaciji virusa SARS-CoV-2. Rezultati so pokazali potencial določenih vzorcev, vendar so za natančnejše informacije o vzorcih potrebne dodatne podrobnejše analize na več različnih tarčah. Knjižnica spojin FKKT trenutno služi predvsem hranjenju vzorcev. Ob nadaljnjem širjenju knjižnice je pričakovana podrobnejša analiza vzorcev v knjižnici. Libraries are the main source of notes, books, information and everything related to information. The development of libraries is a consequence of the development of science. The development of science was accompanied by the development of special libraries, which include chemical compound libraries. Compound storage, quick access to information, easy access to compounds, research on new materials, and research in medicine are just some of the purposes of chemical compound libraries. In 2018, the Faculty of Chemistry and Chemical Technology (FKKT) in Ljubljana opened its own chemical compounds library called "Knjižnica spojin FKKT". The library has a physical and a virtual form. The virtual compound library contains structures and some descriptors of the collected compounds. The FKKT chemical compound library is manually managed and contains compounds synthesized at FKKT. The samples stored in the FKKT chemical compound library are mainly organic. Using NMR analysis, it was determined that 85 % of the samples stored in the FKKT chemical compound library are pure. The remaining 15 % of the samples are contaminated or have decomposed prior to storage. By analyzing the structures from the virtual library, some potential pharmaceutical applications of the compounds were identified. In addition, molecular docking was performed. The biological target of the study was 3CLpro. 3CLpro is an important protease in the replication of the SARS-CoV-2 virus. The analysis provided some promising results. A more detailed analysis would be required to determine the further potential of the FKKT chemical compound library in medicine. Storage of active compounds is currently the primary purpose of the FKKT chemical compound library. In the future, as the number of compounds stored increases, a more detailed compound analysis is expected.

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein., This research was funded by the EuroFlow Consortium which received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA); the grant of the Polish National Center for Research and Development (no. STRATEGMED3/304586/5/NCBR/2017 Person ALL); and internal grant of the Medical University of Silesia (no. PCN-1-050/K/0/K); the grant of CIBER-ONC, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER (no. CB16/12/00400).

The type and degree of contamination on surfaces intended for X-ray photoelectron spectroscopy (XPS) studies is considered decisive for meaningful and reliable analysis as in many cases in-situ cleaning methods are not applicable or otherwise undesired. We report on the effects of sample storage environment on predominantly carbon-and oxygen-containing species accumulating on the surfaces of fourteen types of thin film samples spanning group IVB-VIB transition metals (TMs), TM nitrides, and TM diborides. All specimens were deposited by magnetron sputtering and stored for six months in different common sample storage environments such as openly on a shelf in the office or XPS lab, or within a polypropylene wafer carrier, polystyrene box, cellulose/polyester wipers or sealed polyethylene bag. Self-consistent modelling of C 1s, O 1s, B 1s, N 1s, and metal core level spectra allowed to identify the types and quantities of surface contaminants, metal oxidation states, and thicknesses of native oxides, as well as to address influence from storage ambient type and volume, direct sample contact to other surfaces, or material release from containers. The results reveal significant differences between the various storage types and, hence, provide guidance for all sorts of studies including even those that employ Ar+ ion etch prior to analyses, as also in such cases the amount and type of surface contaminants may impact the outcome. Funding Agencies|Swedish Research Council VR [2018-03957]; Swedish Energy Agency [51201-1]; Carl Tryggers Stiftelse [CTS 20:150, CTS 15:219, CTS 14:431]; Knut and Alice Wallenberg Foundation Scholar [KAW2016.0358, KAW2019.0290]

Biodiversity of soil microbiota is routinely assessed with environmental DNA-based methods, among which amplification and massive parallel sequencing of marker genes (eDNA metabarcoding) is the most common. Soil microbiota may for example be investigated in relation to biodiversity research or as a tool in forensic investigations.After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes after sampling, it is desirable to reduce biological activity, e.g. by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to a questioned item for protracted periods of time.Here we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions.We used five different primer sets targeting bacteria, fungi, protists (cercozoans), general eukaryotes, and plants. For these groups, we quantified differences in richness, evenness and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset.We found increased community composition differences with extended storage time and with higher storage temperature. However, for samples stored less than 28 days at a maximum of 20°C, changes were generally insignificant. Classification models could successfully assign most stored samples to their exact location of origin and correct habitat type even after weeks of storage. Even samples showing larger compositional changes generally retained the original sample as the best match (relative similarity).Our results show that for most biodiversity and forensic applications, storage of samples for days and even several weeks may not be a problem, if storage temperature does not exceed 20°C. Even after suboptimal storage conditions, significant patterns can be reproduced.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder with a birth prevalence of 19.8:100,000 males worldwide. Elevated concentration of the muscle enzyme creatine kinase-MM (CK-MM) allows for presymptomatic screening of newborns using Dried Blood Spots (DBS). We evaluated imprecision and carryover of the FDA-approved PerkinElmer GSP Neonatal CK-MM kit over multiple runs, days, and operators, followed by quantification of CK-MM loss in stored newborn, contrived, and non-newborn patient DBS resulting from exposure to ambient versus low humidity (50-day trial), and high humidity and high temperature (8-day trial). Imprecision %CV was ≤14% for all verification comparisons and over 6 months of testing. On average, the mean CK-MM recovery after 50 days was >80% of initial concentration for all sample types stored in low humidity and

Stability studies are an integral part of the drug development process for any drug product. In addition to monitoring chemical degradation, the physical stability of a drug product must also be evaluated to ensure that the drug release and performance is not affected by storage. In this study, directly compressed tablets of 16 different formulations were exposed to an accelerated stability program to quantify changes in tablet breaking force, porosity, contact angle and disintegration time. Tablets were exposed to five different storage conditions from 37∘C/30% relative humidity (RH) to 70∘C/75%RH with testing after 2 and 4 weeks of storage. Each formulation contained two different fillers (47% w/w each), a disintegrant (5% w/w) and magnesium stearate (1% w/w). The results show that tablets stored at high humidity show increases in porosity and decreases in tensile strength, particularly if they contain a highly hygroscopic filler such as microcrystalline cellulose (MCC). For tablets stored at high temperature, the most commonly affected property was the tablet wettability, measured by sessile drop contact angle measurements. These results are considered in combination with the performance-controlling disintegration mechanism (Maclean et al., 2021) to identify the critical properties which influence the performance after storage., Graphical abstract Unlabelled Image

Storage of biological specimens is crucial in the life and medical sciences. Storage conditions for samples can be different for a number of reasons, and it is unclear what effect this can have on the inferred microbiome composition in metagenomics analyses. Here, we assess the effect of common storage temperatures (deep freezer, −80°C; freezer, −20°C; refrigerator, 5°C; room temperature, 22°C) and storage times (immediate sample processing, 0 h; next day, 16 h; over weekend, 64 h; longer term, 4, 8, and 12 months) as well as repeated sample freezing and thawing (2 to 4 freeze-thaw cycles). We examined two different pig feces and sewage samples, unspiked and spiked with a mock community, in triplicate, respectively, amounting to a total of 438 samples (777 Gbp; 5.1 billion reads). Storage conditions had a significant and systematic effect on the taxonomic and functional composition of microbiomes. Distinct microbial taxa and antimicrobial resistance classes were, in some situations, similarly affected across samples, while others were not, suggesting an impact of individual inherent sample characteristics. With an increasing number of freeze-thaw cycles, an increasing abundance of Firmicutes, Actinobacteria, and eukaryotic microorganisms was observed. We provide recommendations for sample storage and strongly suggest including more detailed information in the metadata together with the DNA sequencing data in public repositories to better facilitate meta-analyses and reproducibility of findings. IMPORTANCE Previous research has reported effects of DNA isolation, library preparation, and sequencing technology on metagenomics-based microbiome composition; however, the effect of biospecimen storage conditions has not been thoroughly assessed. We examined the effect of common sample storage conditions on metagenomics-based microbiome composition and found significant and, in part, systematic effects. Repeated freeze-thaw cycles could be used to improve the detection of microorganisms with more rigid cell walls, including parasites. We provide a data set that could also be used for benchmarking algorithms to identify and correct for unwanted batch effects. Overall, the findings suggest that all samples of a microbiome study should be stored in the same way. Furthermore, there is a need to mandate more detailed information about sample storage and processing be published together with DNA sequencing data at the International Nucleotide Sequence Database Collaboration (ENA/EBI, NCBI, DDBJ) or other repositories.

Nasr Alrabadi,1 Hanin Makhlouf,2 Omar F Khabour,2 Karem H Alzoubi3 1Department of Pharmacology, Faculty Of Medicine, Jordan University of Science and Technology, Irbid, Jordan; 2Department of Medical Laboratory Sciences, Faculty of Medical Sciences, Jordan University of Science and Technology, Irbid, Jordan; 3Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, JordanCorrespondence: Nasr AlrabadiDepartment Of Pharmacology, Faculty Of Medicine, Jordan University Of Science And Technology, Irbid 22110, JordanTel +962795994247Email nnalrabadi@just.edu.joIntroduction: The informed consent process is an integral step in biomedical research. However, the emergence of biobanks and the need for open consent (also called “broad” or “blanket” consent) create challenges to this process.Aims and methodology: A survey was used to examine Jordanians’ perspectives on open consent and reuse of stored samples in future research.Results: The majority of participants had positive perceptions of informed consent and its importance. In addition, they appreciated the challenges that are associated with multiple uses of their biospecimens. About 55% agreed to provide open consent for reuse of their donated biospecimens. Participants (75–80%) also agreed that issues such as the possibility of sharing samples with international research centers, storage duration, and use of biospecimens after their death should be clarified as part of open consent. The inconvenience of the re-contact process, trust in the research team, and the importance of biobanks were all associated with participants’ willingness to provide open consent (P

Hanin Makhlouf,1 Nasr Alrabadi,2 Omar F Khabour,1 Karem H Alzoubi,3 Wael Al-Delaimy4 1Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan; 2Department of Pharmacology, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan; 3Department of Clinical Pharmacy, Jordan University of Science and Technology, Irbid, Jordan; 4Division of Global Health, Department of Family and Preventive Medicine, University of California at San Diego, San Diego, CA, USA Background: Biobanks (biorepositories) were established to compile collected bio-specimens for future research and usage. The collection/storage of bio-specimens triggers several social, legal, and ethical implications where public attitudes can represent the core measurement/parameter in defining the most acceptable practices and ethical approaches when dealing with biobanks. Aim: The aim of this study was to explore and understand population’s perspectives, expectations, and concerns toward biobanks in Jordan. Methods: A cross-sectional survey that included closed-ended questions was distributed among Jordanians. A total of 500 participants who are representative of the Jordanian population were included in this study. Results: There was overwhelming support (>85%) for the establishment of biobanks in Jordan, and most of the participants agreed on the importance of biobanks and samples’ donation for promoting medical research. Enthusiasm in biobanking participation was associated with the sociodemographic characteristics of participants including age, educational level, and previous knowledge of biobanks. Moreover, considering sample donation as a religiously good deed appeared to have the strongest positive correlation with willingness to donate bio-specimens for future research. Also, participants’ trust in medical and research services, especially the protection of their privacy and confidentiality, was the most critical concern when they decided to participate in biobanks. Conclusion: Population’s attitude toward biobanks in Jordan was positive and promising, and can encourage the future establishment of different biobanks. It is also necessary to take into consideration certain sociodemographic characteristics when discussing specific information with potential biobanks’ donors. Keywords: biorepositories, sample storage, sample donation, sample reuse, ethics

Phosphomonoesterases play an important role in the soil phosphorus (P) cycle since they hydrolyze P monoester to phosphate. Their activity is generally measured in soil extracts, and thus, it remains uncertain how mobile these enzymes are and to which extent they can be translocated within the soil profile. The presence of phosphomonoesterases in soil solutions potentially affects the share of labile dissolved organic P (DOP), which in turn would affect P leaching. Our study aimed at assessing the production and leaching of phosphomonoesterases from organic layers and topsoil horizons in forest soils and its potential effect on dissolved P forms in leachates obtained from zero-tension lysimeters. We measured phosphomonoesterase activities in leached soil solutions and compared it with those in water extracts from litter, Oe/Oa, and A horizons of two beech forests with a contrasting nitrogen (N) and P availability, subjected to experimental N × P fertilization. In addition, we determined phosphate and DOP. In soil solutions leached from litter, Oe/Oa, and A horizons, phosphomonoesterase activities ranged from 2 to 8 μmol L–1 h–1 during summer, but remained below detection limits in winter. The summer values represent 0.1–1% of the phosphomonoesterase activity in soil extracts, indicating that enzymes can be translocated from organic layers and topsoils to greater soil depths. Activities of phosphomonoesterases obtained by water extracts were greater in the organic layer of the P-poor site, while activities of those in soil solutions were similar at the two sites. Nitrogen addition increased phosphomonoesterase activities in leached soil solutions of the organic layer of the N- and P-poor soil. Using a modeling approach, we estimated that approx. 76% of the initial labile DOP was hydrolyzed to dissolved inorganic P within the first 24 h. Back calculations from measured labile DOP revealed an underestimation of approx. 15% of total dissolved P, or 0.03 mg L–1. The observed leaching of phosphomonoesterases implies that labile organic P could be hydrolyzed in deeper soil horizons and that extended sample storage leads to an underestimation of the contribution of DOP to total dissolved P leaching. This has been neglected in the few field studies measuring DOP leaching.

Congenital cytomegalovirus (cCMV) infection is a leading non-genetic cause of sensorineural hearing. Alethia CMV assay is proved to be accurate, simple and appears suitable for CMV testing using neonatal saliva. However, long storage of swab samples is not validated across different time periods and storage temperature. This study verifies the effects of storage time and temperature in detection of CMV from saliva samples using molecular assay developed by Alethia.

Understanding the roles of microorganisms in the animal gastrointestinal microenvironment is highly important for the development of effective strategies to manage and manipulate these microbial communities. In order to guide future animal gut microbiota research projects and standardization efforts, we have conducted a systematic comparison of 10 currently used sample preservation and DNA extraction approaches for pig and chicken microbiota samples and quantified their effects on bacterial DNA yield, quality, integrity, and on the resulting sequence-based bacterial composition estimates. The results showed how key stages of conducting a microbiota study, including the sample storage and DNA extraction, can substantially affect DNA recovery from the microbial community, and therefore, biological interpretation in a matrix-dependent manner. Our results highlight the fact that the influence of storage and extraction methods on the resulting microbial community structure differed by sample type, even within the same species. As the effects of these technical steps are potentially large compared with the real biological variability to be explained, standardization is crucial for accelerating progress in the area of livestock microbiota research. This study provided a framework to assist future animal gut microbiota research projects and standardization efforts.

Background Quality of the nucleic acids extracted from Formalin Fixed Paraffin Embedded (FFPE) samples largely depends on pre-analytic, fixation and storage conditions. We assessed the differential sensitivity of viral and human double stranded DNA (dsDNA) to degradation with storage time. Methods We randomly selected forty-four HPV16-positive invasive cervical cancer (ICC) FFPE samples collected between 1930 and 1935 and between 2000 and 2004. We evaluated through qPCR the amplification within the same sample of two targets of the HPV16 L1 gene (69 bp, 134 bp) compared with two targets of the human tubulin-β gene (65 bp, 149 bp). Results Both viral and human, short and long targets were amplified from all samples stored for 15 years. In samples archived for 85 years, we observed a significant decrease in the ability to amplify longer targets and this difference was larger in human than in viral DNA: longer fragments were nine times (CI 95% 2.6–35.2) less likely to be recovered from human DNA compared with 1.6 times (CI 95% 1.1–2.2) for viral DNA. Conclusions We conclude that human and viral DNA show a differential decay kinetics in FFPE samples. The faster degradation of human DNA should be considered when assessing viral DNA prevalence in long stored samples, as HPV DNA detection remains a key biomarker of viral-associated transformation.

Dried blood spot (DBS) technology has become a promising utility for the transportation and storage of biological fluids aimed for the subsequent clinical analysis. The basis of the DBS method is the adsorption of the components of a biological sample onto the surface of a membrane carrier, followed by drying. After drying, the molecular components of the biosample (nucleic acids, proteins, and metabolites) can be analyzed using modern omics, immunological, or genomic methods. In this work, we investigated the safety of proteins on a membrane carrier by tryptic components over time and at different temperatures (+4, 0, 25 &deg, C) and storage (0, 7, 14, and 35 days). It was shown that the choice of a protocol for preliminary sample preparation for subsequent analytical molecular measurements affects the quality of the experimental results. The protocol for preliminary preparation of a biosample directly in a membrane carrier is preferable compared to the protocol with an additional stage of elution of molecular components before the sample preparation procedures. It was revealed that the composition of biosamples remains stable at a temperature of &minus, 20 and +4 &deg, C for 35 days of storage, and at +25 &deg, C for 14 days.

Quantitative nuclear magnetic resonance (NMR) spectroscopy of blood plasma is widely used to investigate perturbed metabolic processes in human diseases. The reliability of biochemical data derived from these measurements is dependent on the quality of the sample collection and exact preparation and analysis protocols. Here, we describe systematically, the impact of variations in sample collection and preparation on information recovery from quantitative proton (1H) NMR spectroscopy of human blood plasma and serum. The effects of variation of blood collection tube sizes and preservatives, successive freeze-thaw cycles, sample storage at -80 °C, and short-term storage at 4 and 20 °C on the quantitative lipoprotein and metabolite patterns were investigated. Storage of plasma samples at 4 °C for up to 48 h, freezing at -80 °C and blood sample collection tube choice have few and minor effects on quantitative lipoprotein profiles, and even storage at 4 °C for up to 168 h caused little information loss. In contrast, the impact of heat-treatment (56 °C for 30 min), which has been used for inactivation of SARS-CoV-2 and other viruses, that may be required prior to analytical measurements in low level biosecurity facilities induced marked changes in both lipoprotein and low molecular weight metabolite profiles. It was conclusively demonstrated that this heat inactivation procedure degrades lipoproteins and changes metabolic information in complex ways. Plasma from control individuals and SARS-CoV-2 infected patients are differentially altered resulting in the creation of artifactual pseudo-biomarkers and destruction of real biomarkers to the extent that data from heat-treated samples are largely uninterpretable. We also present several simple blood sample handling recommendations for optimal NMR-based biomarker discovery investigations in SARS CoV-2 studies and general clinical biomarker research.

The impact of storage temperature (4, 22 and 37 &deg, C) and storage time (7, 14 and 21 days) on anaerobic digestion inocula was investigated through specific methanogenic activity assays. Experimental results showed that methanogenic activity decreased over time with storage, regardless of storage temperature. However, the rate at which the methanogenic activity decreased was two and five times slower at 4 &deg, C than at 22 and 37 &deg, C, respectively. The inoculum stored at 4 &deg, C and room temperature (22 &deg, C) maintained methanogenic activity close to that of fresh inoculum for 14 days (&lt, 10% difference). However, a storage temperature of 4 &deg, C is preferred because of the slower decrease in activity with lengthier storage time. From this research, it was concluded that inoculum storage time should generally be kept to a minimum, but that storage at 4 &deg, C could help maintain methanogenic activity for longer.

The WHO-named Coronavirus Disease 2019 (COVID-19) infection had become a pandemic within a short time period since it was detected in Wuhan. The outbreak required the screening of millions of samples daily and overwhelmed diagnostic laboratories worldwide. During this pandemic, the handling of patient specimens according to the universal guidelines was extremely difficult as the WHO, CDC and ECDC required cold chain compliance during transport and storage of the swab samples. The aim of this study was to compare the effects of two different storage conditions on the COVID-19 real-time PCR assay on 30 positive nasopharyngeal and/or oropharyngeal samples stored at both ambient temperature (22 ± 2 °C) and +4 °C. The results revealed that all the samples stored at ambient temperature remain PCR positive for at least six days without any false-negative result. In conclusion, transporting and storing these types of swab samples at ambient temperature for six days under resource-limited conditions during the COVID-19 pandemics are acceptable.

BackgroundWell-characterized biomaterials of high quality have great potential for acceleration and quality improvement in translational biomedical research. To improve accessibility of local sample collections, efforts have been made to create central biomaterial banks and catalogues. Available technical solutions for creating professional local sample catalogues and connecting them to central systems are cost intensive and/or technically complex to implement. Therefore, the Translational Thematic Unit HIV of the German Center for Infection Research (DZIF) developed a Laboratory Information and Management System (LIMS) called HIV Engaged Research Technology (HEnRY) for implementation into the Translational Platform HIV (TP-HIV) at the DZIF and other research networks.ResultsHEnRY is developed at the University Hospital of Cologne. It is an advanced LIMS to manage processing and storage of samples and aliquots of different sample types. Features include:monitoring of stored samples and associated informationdata selection via query tools or Structured Query Language (SQL)preparation of summary documents, including scannable search listscentralized management of the practical laboratory part of multicentre studies (e.g. import of drawing schemes and sample processing steps),preparation of aliquot shipments, including associated documents to be added to shipmentsunique and secure identification of aliquots through use of customizable Quick Response (QR) code labels directly from HEnRYsupport of aliquot data transmission to central registries.In summary, HEnRY offers all features necessary for a LIMS software. In addition, the structure of HEnRY provides sufficient flexibility to allow the implementation in other research areas.ConclusionHEnRY is a free biobanking tool published under the MIT license. While it was developed to support HIV research in Germany, the feature set and language options, allow much broader applications and make this a powerful free research tool.

The low reproducibility of biomarker research is a major holdback for the translation of research results to the bedside. Sample integrity has been identified as a key factor that contributes to improved reproducibility. The key mission of biobanks is to ensure that all activities and materials are managed according to standardized procedures and best practices to ensure and preserve sample integrity. When handling large numbers of biospecimens automation of sample handling and storage is often the method of choice to maintain and improve sample integrity. In December 2013, the centralized Biobank of the University Hospitals and the Catholic University of Leuven (UZ KU Leuven) decided to implement automated systems for sample storage and retrieval, one for storage at -20°C and one for storage at -80°C. Here we describe the extensive process of installation, acceptance, validation, and implementation of these two systems. Overall it took about 4 years to effectively take the systems into production. Multiple issues resulted in the delayed implementation, with labware change, quality of the initial installation, and misunderstanding of biobank concerns being the most impacting. Significant effort in terms of time and resources from both the automated store supplier as well as the biobank itself was needed to achieve a successful implementation. Within 15 months of actual integration in the biobank workflow, over 63 k samples were placed into the systems. Actual hands-on sample handling and retrieval times were substantially reduced, although this implied the shift of dedicated personnel time from the researchers' laboratories to the biobank. With the successful implementation of automated frozen sample storage systems, the centralized UZ KU Leuven Biobank is now also able to efficiently support large-scale translational research. ispartof: FRONTIERS IN MEDICINE vol:6 ispartof: location:Switzerland status: published

Acute otitis media (AOM) is the most common pediatric infection for which antibiotics are prescribed in the United States. The role of the respiratory tract microbiome in pathogenesis and immune modulation of AOM remains unexplored. We sought to compare the nasopharyngeal (NP) microbiome of children 1 to 3 weeks prior to onset of AOM vs. at onset of AOM, and the NP microbiome with the microbiome in middle ear (ME). Six children age 6 to 24 months old were studied. Nasal washes (NW) were collected at healthy visits 1 to 3 weeks prior to AOM and at onset of AOM. The middle ear fluids (MEF) were collected by tympanocentesis at onset of AOM. Samples were stored in Trizol reagents or phosphate-buffered saline (PBS) at −80°C until use. The microbiome was characterized by 16S rRNA gene sequencing. Taxonomic designations and relative abundance of bacteria were determined using the RDP classifier tool through QIIME. Cumulative sum scaling normalization was applied before determining bacterial diversity and abundance. Shannon diversity index was calculated in Microsoft excel. The relative abundance of each bacteria species was compared via Mann-Whitney U test. We found that the NW microbiome of children during healthy state or at baseline was more diverse than microbiome during AOM. At AOM, no significant difference in microbiome diversity was found between NW and MEF, although some bacteria species appear to differ in MEF than in NW. The microbiome of samples stored in PBS had significant greater diversity than samples stored in Trizol reagent.

With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at &minus, 80 &deg, C should be chosen wherever possible.

Sample storage for downstream RNA analysis can be challenging in some field settings, especially where access to cryogenic materials or refrigeration/freezer facilities are limited. This has limited RNA-based studies on African malaria vectors collected in the field. We evaluated RNA quality after storing mosquito samples in three different sample preservation media over a 4-week period. Storing mosquito specimens in cold (4°C) media significantly improved yields of intact RNA. Our results indicate commercially available products perform well in keeping RNA integrity as advertised. Moreover, absolute ethanol may be an economical alternative for sample preservation that can be utilized in some resource-limited settings.

Introduction The informed consent process is an integral step in biomedical research. However, the emergence of biobanks and the need for open consent (also called “broad” or “blanket” consent) create challenges to this process. Aims and methodology A survey was used to examine Jordanians’ perspectives on open consent and reuse of stored samples in future research. Results The majority of participants had positive perceptions of informed consent and its importance. In addition, they appreciated the challenges that are associated with multiple uses of their biospecimens. About 55% agreed to provide open consent for reuse of their donated biospecimens. Participants (75–80%) also agreed that issues such as the possibility of sharing samples with international research centers, storage duration, and use of biospecimens after their death should be clarified as part of open consent. The inconvenience of the re-contact process, trust in the research team, and the importance of biobanks were all associated with participants’ willingness to provide open consent (P

Background Biobanks (biorepositories) were established to compile collected bio-specimens for future research and usage. The collection/storage of bio-specimens triggers several social, legal, and ethical implications where public attitudes can represent the core measurement/parameter in defining the most acceptable practices and ethical approaches when dealing with biobanks. Aim The aim of this study was to explore and understand population’s perspectives, expectations, and concerns toward biobanks in Jordan. Methods A cross-sectional survey that included closed-ended questions was distributed among Jordanians. A total of 500 participants who are representative of the Jordanian population were included in this study. Results There was overwhelming support (>85%) for the establishment of biobanks in Jordan, and most of the participants agreed on the importance of biobanks and samples’ donation for promoting medical research. Enthusiasm in biobanking participation was associated with the sociodemographic characteristics of participants including age, educational level, and previous knowledge of biobanks. Moreover, considering sample donation as a religiously good deed appeared to have the strongest positive correlation with willingness to donate bio-specimens for future research. Also, participants’ trust in medical and research services, especially the protection of their privacy and confidentiality, was the most critical concern when they decided to participate in biobanks. Conclusion Population’s attitude toward biobanks in Jordan was positive and promising, and can encourage the future establishment of different biobanks. It is also necessary to take into consideration certain sociodemographic characteristics when discussing specific information with potential biobanks’ donors.

BackgroundBiobanks (biorepositories) were established to compile collected bio-specimens for future research and usage. The collection/storage of bio-specimens triggers several social, legal, and ethical implications where public attitudes can represent the core measurement/parameter in defining the most acceptable practices and ethical approaches when dealing with biobanks.AimThe aim of this study was to explore and understand population's perspectives, expectations, and concerns toward biobanks in Jordan.MethodsA cross-sectional survey that included closed-ended questions was distributed among Jordanians. A total of 500 participants who are representative of the Jordanian population were included in this study.ResultsThere was overwhelming support (>85%) for the establishment of biobanks in Jordan, and most of the participants agreed on the importance of biobanks and samples' donation for promoting medical research. Enthusiasm in biobanking participation was associated with the sociodemographic characteristics of participants including age, educational level, and previous knowledge of biobanks. Moreover, considering sample donation as a religiously good deed appeared to have the strongest positive correlation with willingness to donate bio-specimens for future research. Also, participants' trust in medical and research services, especially the protection of their privacy and confidentiality, was the most critical concern when they decided to participate in biobanks.ConclusionPopulation's attitude toward biobanks in Jordan was positive and promising, and can encourage the future establishment of different biobanks. It is also necessary to take into consideration certain sociodemographic characteristics when discussing specific information with potential biobanks' donors.

As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout (Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at − 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses. peerReviewed

Background A majority of indoor residential microbes originate from humans, pets, and outdoor air and are not adapted to the built environment (BE). Consequently, a large portion of the microbes identified by DNA-based methods are either dead or metabolically inactive. Although many exceptions have been noted, the ribosomal RNA fraction of the sample is more likely to represent either viable or metabolically active cells. We examined methodological variations in sample processing using a defined, mock BE microbial community to better understand the scope of technique-based vs. biological-based differences in both ribosomal transcript (rRNA) and gene (DNA) sequence community analysis. Based on in vitro tests, a protocol was adopted for the analysis of the genetic and metabolic pool (DNA vs. rRNA) of air and surface microbiomes within a residential setting. Results We observed differences in DNA/RNA co-extraction efficiency for individual microbes, but overall, a greater recovery of rRNA using FastPrep (> 50%). Samples stored with various preservation methods at − 80°C experienced a rapid decline in nucleic acid recovery starting within the first week, although post-extraction rRNA had no significant degradation when treated with RNAStable. We recommend that co-extraction samples be processed as quickly as possible after collection. The in vivo analysis revealed significant differences in the two components (genetic and metabolic pool) in terms of taxonomy, community structure, and microbial association networks. Rare taxa present in the genetic pool showed higher metabolic potential (RNA:DNA ratio), whereas commonly detected taxa of outdoor origins based on DNA sequencing, especially taxa of the Sphingomonadales order, were present in lower relative abundances in the viable community. Conclusions Although methodological variations in sample preparations are high, large differences between the DNA and RNA fractions of the total microbial community demonstrate that direct examination of rRNA isolated from a residential BE microbiome has the potential to identify the more likely viable or active portion of the microbial community. In an environment that has primarily dead and metabolically inactive cells, we suggest that the rRNA fraction of BE samples is capable of providing a more ecologically relevant insight into the factors that drive indoor microbial community dynamics. Electronic supplementary material The online version of this article (10.1186/s40168-018-0453-0) contains supplementary material, which is available to authorized users.

Introduction The aim of the study was to evaluate the effect of a single freeze/thaw cycle on HbA1c concentrations measured by commercially available HPLC method. Materials and methods Study included 128 whole blood samples collected from diabetic patients (N = 60) and healthy volunteers (N = 68). HbA1c concentrations were measured in fresh blood samples. Then samples were frozen at - 80 °C for up to 12 weeks. HbA1c was assayed by ion-exchange HPLC method on Bio-Rad D-10® analyzer. Variables were compared using Wilcoxon and ANOVA Kruskal-Wallis tests. Bias between HbA1c measured in fresh and frozen samples was calculated. The comparability of HbA1c concentrations was assessed by Bland-Altman plot. Results Median (IQR) HbA1c concentration was 45.3 (36.6-61.2) mmol/mol for fresh and 45.3 (36.6-60.6) mmol/mol for frozen/thawed samples. No significant difference in HbA1c concentrations was found comparing fresh and frozen/thawed samples (P = 0.070) in the whole group, as well as in healthy and diabetic subjects. The median calculated bias between fresh and frozen/thawed samples was 0% in whole group and healthy subjects, and 1.19% in diabetic patients. No significant difference was found between the biases according to baseline HbA1c values (P = 0.150). The Bland-Altman plot analysis showed a positive bias of 0.4% (95% CI: - 2.8 - 3.7%), which indicates high compliance between HbA1c values and no relevant influence of sample freezing on clinical significance of HbA1c measurement. Conclusions Storage for up to 12 weeks at - 80 °C with a single freeze/thaw cycle does not affect HbA1c concentrations measured with HPLC method on Bio-Rad D-10® analyzer.

Background The increasing interest on the impact of the gut microbiota on health and disease has resulted in multiple human microbiome-related studies emerging. However, multiple sampling methods are being used, making cross-comparison of results difficult. To avoid additional clinic visits and increase patient recruitment to these studies, there is the potential to utilize at-home stool sampling. The aim of this pilot study was to compare simple self-sampling collection and storage methods. Methods To simulate storage conditions, stool samples from three volunteers were freshly collected, placed on toilet tissue, and stored at four temperatures (− 80, 7, 22 and 37 °C), either dry or in the presence of a stabilization agent (RNAlater®) for 3 or 7 days. Using 16S rRNA gene sequencing by Illumina, the effect of storage variations for each sample was compared to a reference community from fresh, unstored counterparts. Fastq files may be accessed in the NCBI Sequence Read Archive: Bioproject ID PRJNA418287. Results Microbial diversity and composition were not significantly altered by any storage method. Samples were always separable based on participant, regardless of storage method suggesting there was no need for sample preservation by a stabilization agent. Discussion In summary, if immediate sample processing is not feasible, short term storage of unpreserved stool samples on toilet paper offers a reliable way to assess the microbiota composition by 16S rRNA gene sequencing.

Metabolomics has great potential for uncovering biomarkers of the disease state in CF and many other contexts. However, sample storage timing and temperature may alter the abundance of clinically relevant metabolites. To assess whether existing samples are stable and to direct future study design, we conducted untargeted GC-MS metabolomic analysis of CF sputum samples after one or two freeze-thaw cycles and storage at 4°C and −20°C for 4 to 8 weeks. Overall, storage at −20°C and freeze-thaw cycles had little impact on metabolite profiles; however, storage at 4°C shifted metabolite abundances significantly. GC-MS profiling will aid in our understanding of the CF lung, but care should be taken in studies using sputum samples to ensure that samples are properly stored., Metabolites of human or microbial origin have the potential to be important biomarkers of the disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography-mass spectrometry (GC-MS) when CF sputum samples were stored at 4°C, −20°C, or −80°C with one or two freeze-thaw cycles. Daily measurements were taken for 1 week and then weekly for 4 weeks (4°C) and 8 weeks (−20°C). The metabolites in samples stored at −20°C maintained abundances similar to those found at−80°C over the course of 8 weeks (average change in Bray-Curtis distance, 0.06 ± 0.04) and were also stable after one or two freeze-thaw cycles. However, the metabolite profiles of samples stored at 4°C shifted after 1 day and continued to change over the course of 4 weeks (average change in Bray-Curtis distance, 0.31 ± 0.12). The abundances of several amino acids and other metabolites increased with time of storage at 4°C but remained constant at −20°C. Storage temperature was a significant factor driving the metabolite composition (permutational multivariate analysis of variance: r2 = 0.32 to 0.49, P < 0.001). CF sputum samples stored at −20°C at the time of sampling maintain a relatively stable untargeted GC-MS profile. Samples should be frozen on the day of collection, as more than 1 day at 4°C impacts the global composition of the metabolites in the sample. IMPORTANCE Metabolomics has great potential for uncovering biomarkers of the disease state in CF and many other contexts. However, sample storage timing and temperature may alter the abundance of clinically relevant metabolites. To assess whether existing samples are stable and to direct future study design, we conducted untargeted GC-MS metabolomic analysis of CF sputum samples after one or two freeze-thaw cycles and storage at 4°C and −20°C for 4 to 8 weeks. Overall, storage at −20°C and freeze-thaw cycles had little impact on metabolite profiles; however, storage at 4°C shifted metabolite abundances significantly. GC-MS profiling will aid in our understanding of the CF lung, but care should be taken in studies using sputum samples to ensure that samples are properly stored.

Many metabolomics studies use mixtures of (acidified) methanol and water for sample extraction. In the present study, we investigated if the extraction with methanol can result in artifacts. To this end, wheat leaves were extracted with mixtures of native and deuterium-labeled methanol and water, with or without 0.1% formic acid. Subsequently, the extracts were analyzed immediately or after storage at 10 °C, −20 °C or −80 °C with an HPLC-HESI-QExactive HF-Orbitrap instrument. Our results showed that 88 (8%) of the >1100 detected compounds were derived from the reaction with methanol and either formed during sample extraction or short-term storage. Artifacts were found for various substance classes such as flavonoids, carotenoids, tetrapyrrols, fatty acids and other carboxylic acids that are typically investigated in metabolomics studies. 58 of 88 artifacts were common between the two tested extraction variants. Remarkably, 34 of 73 (acidified extraction solvent) and 33 of 73 (non-acidified extraction solvent) artifacts were formed de novo as none of these meth(ox)ylated metabolites were found after extraction of native leaf samples with CD3OH/H2O. Moreover, sample extracts stored at 10 °C for several days, as can typically be the case during longer measurement sequences, led to an increase in both the number and abundance of methylated artifacts. In contrast, frozen sample extracts were relatively stable during a storage period of one week. Our study shows that caution has to be exercised if methanol is used as the extraction solvent as the detected metabolites might be artifacts rather than natural constituents of the biological system. In addition, we recommend storing sample extracts in deep freezers immediately after extraction until measurement.

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P

Fluvial fluxes of dissolved organic carbon (DOC) may represent an important loss for terrestrial carbon stores in the tropics. However, there is currently limited guidance on the preservation of tropical water samples for DOC analysis. Commonly employed preservation techniques such as freezing or acidification can limit degradation but may also alter sample properties, complicating DOC analysis. We examined the effects of cold storage at 4 °C on DOC concentration and quality in water samples collected from a tropical peat catchment. Samples were stored in the dark at 4 °C for periods of 6–12 weeks. Freeze/thaw experiments were also made. Mean DOC concentrations in samples stored for six weeks at 4 °C were 6.1 % greater than in samples stored at ambient room temperature (33 °C) over the same period. Changes in DOC concentrations, in two sample sets, during cold storage were 2.25 ± 2.9 mg L-1 (8 %) to 2.69 ± 1.4 mg L-1 (11 %) over a 12-week period. Freeze/thaw resulted in alterations in the optical properties of samples, and this in turn altered the calculated DOC concentrations by an average of 10.9 %. We conclude that cold storage at 4 °C is an acceptable preservation method for tropical DOC water samples, for moderate time periods, and is preferable to freezing or storage at ambient temperatures.

It is not uncommon for research and quality control samples, including carbonated beverage samples, to be refrigerated or frozen during peak periods of production and/or sampling, when analytical demand exceeds instrumental capacity. However, the effect of sub-ambient temperatures on carbonated beverage composition during storage has not been well characterized. Mid-infrared(MIR) spectroscopy combined with principal component analysis (PCA) and traditional chemical analyses were used to evaluate the effects of refrigeration (for 1 week) and freezing (for 1 or 6 weeks)on the composition of carbonated beverages, including sparkling water, sparkling wine, beer,and cider. Carbonated beverages were generally resistant to changes in pH, titratable acidity,alcohol, total phenolics, sugar, and color, during short-term (1 week) storage. However, long-term(6 week) freezing resulted in decreased total phenolics, with acidity also affected, albeit to a lesser extent. MIR spectroscopy combined with PCA enabled discrimination of carbonated beverages based on composition, with alcohol content having a significant influence. Examination of theMIR ‘fingerprint’ region indicated subtle compositional changes occurred in carbonated beveragesfollowing prolonged freezing. Refereed/Peer-reviewed

It is not uncommon for research and quality control samples, including carbonated beverage samples, to be refrigerated or frozen during peak periods of production and/or sampling, when analytical demand exceeds instrumental capacity. However, the effect of sub‐ambient temperatures on carbonated beverage composition during storage has not been well characterized. Mid‐infrared (MIR) spectroscopy combined with principal component analysis (PCA) and traditional chemical analyses were used to evaluate the effects of refrigeration (for 1 week) and freezing (for 1 or 6 weeks) on the composition of carbonated beverages, including sparkling water, sparkling wine, beer, and cider. Carbonated beverages were generally resistant to changes in pH, titratable acidity, alcohol, total phenolics, sugar, and color, during short‐term (1 week) storage. However, long‐term (6 week) freezing resulted in decreased total phenolics, with acidity also affected, albeit to a lesser extent. MIR spectroscopy combined with PCA enabled discrimination of carbonated beverages based on composition, with alcohol content having a significant influence. Examination of the MIR ‘fingerprint’ region indicated subtle compositional changes occurred in carbonated beverages following prolonged freezing.

The reliability and accuracy of blood alcohol concentration (BAC) results presented in South African courts of law in respect of possible driving under the influence (DUI) cases, have in recent years been subjected to intense scrutiny and severe criticism. Research has shown that multiple factors may negatively impact on the reliability of results obtained from the analysis of such samples - including inappropriate or non-standardised sample management after acquisition thereof. In particular, long delays between sample acquisition and the analysis thereof may compromise the validity of results. Such delays may also negatively affect the outcome of both criminal and civil legal proceedings in possible DUI cases. A retrospective descriptive study was conducted on records from the Pretoria Forensic Chemistry Laboratory (PFCL) regarding the relevant dates pertaining to blood samples from deceased persons which were received for analysis. These parameters included the date of sample acquisition at medico-legal mortuaries, delays in submission of samples to the laboratory and date of actual analysis. In addition, the expiration dates of sample collection kits were recorded. Our results show that numerous expired kits were utilised and that there was an average delay of approximately five months between sample acquisition and laboratory analysis thereof. This delay period varied greatly but appears to correlate with geographical distance of the medico-legal mortuary from the PFCL. In order to optimise and facilitate the administration of justice in both criminal and civil cases of alleged DUI, these shortcomings should be urgently addressed. It is argued that the implementation of prescribed measures and standard operating procedures in sample management, together with other interventions such as accreditation of laboratories and improved resourcing of medico-legal and toxicology laboratories, is urgently required.

BackgroundPreanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples.MethodsPooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min.ResultsWithin treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice.DiscussionCanine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice-water bath rather than in ice. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality.

Enzymeimmunoassays (EIAs) allow researchers to monitor stress hormone output via measurement of fecal glucocorticoid metabolites (FGCMs) in many vertebrates. They can be powerful tools which allow the acquisition of otherwise unobtainable physiological information from both captive animals and wild animals in remote forest habitats, such as great apes. However, methods for hormone measurement, extraction and preservation need to be adapted and validated for field settings. In preparation for a field study of Western lowland gorillas (Gorilla gorilla gorilla) in the Central African Republic we used samples from captive gorillas collected around opportunistic stressful situations to test whether four different glucocorticoid EIAs reflected adrenocortical activity reliably and to establish the lag-time from the stressor to peak excretion. We also validated a field extraction technique and established a simple, non-freezer-reliant method to preserve FGCMs in extracts long-term. We determined the rate of FGCM change over 28 days when samples cannot be extracted immediately and over 12 h when feces cannot be preserved immediately in alcohol. Finally, we used repeat samples from identified individuals to test for diurnal variation in FGCM output. Two group-specific assays measuring major cortisol metabolites detected the predicted FGCM response to the stressor reliably, whereas more specific cortisol and corticosterone assays were distinctly less responsive and thus less useful. We detected a lag time of 2–3 days from stressor to peak FGCM excretion. Our field extraction method performed as well as an established laboratory extraction method and FGCMs in dried extracts stored at ambient temperatures were as stable as those at −20 °C over 1 yr. Hormones in non-extracted feces in alcohol were stable up to 28 days at ambient temperatures. FGCMs in un-fixed gorilla feces deteriorated to almost 50% of the original values within 6 h under field conditions. We detected no diurnal variation in FGCMs in samples from wild gorillas. Our study highlights the importance of thorough biological and immunological validation of FGCM assays, and presents validated, practical methods for the application of non-invasive adrenocortical monitoring techniques to field conservation contexts where it is crucially needed.

Stools are commonly used as proxies for studying human gut microbial communities as sample collection is straightforward, cheap and non-invasive. In large-scale human population surveys, however, sample integrity becomes an issue as it is not logistically feasible for researchers to personally collect stools from every participant. Instead, participants are usually given guidelines on sample packaging and storage, and asked to deliver their stools to a centralised facility. Here, we tested a number of delivery conditions (temperature, duration and addition of preservative medium) and assessed their effects on stool microbial community composition using 16S rRNA gene amplicon sequencing. The largest source of variability in stool community composition was attributable to inter-individual differences regardless of delivery condition. Although the relative effect of delivery condition on community composition was small compared to inter-individual variability (1.6% vs. 60.5%, permutational multivariate analysis of variance [PERMANOVA]) and temporal variation within subjects over 10 weeks (5.2%), shifts in microbial taxa associated with delivery conditions were non-systematic and subject-specific. These findings indicated that it is not possible to model or accurately predict shifts in stool community composition associated with sampling logistics. Based on our findings, we recommend delivery of fresh, preservative-free stool samples to laboratories within 2 hr either at ambient or chilled temperatures to minimise perturbations to microbial community composition. In addition, subsamples from different fractions of the same stool displayed a small (3.3% vs. 72.6% inter-individual variation, PERMANOVA) but significant effect on community composition. Collection of larger sample volumes for homogenisation is recommended.

The changes of ethanol concentrations in whole blood and urine samples were analyzed depending on temperatures and duration of storage. The aim of the study was to establish standards for the Institute laboratory. Samples of whole blood and urine, taken from drivers with excessive alcohol concentrations (6 groups, 15 samples per each), were analyzed upon delivery and then after storage during different time intervals and at different temperatures. The results showed that alcohol concentrations were significantly reduced with the increase of temperature and prolongation of storage. Only the whole blood samples stored for up to one month at -20°C did not show significant changes. Room temperature storage of samples is the least suitable way of keeping them, independently of the duration of storage. Urines are not less reliable samples than blood. There are no ethanol differences between blood samples with and without sodium fluoride.

Background A review of the literature revealed limited information about the stability of samples for coagulation testing in dogs. Objective The aim of this study was to evaluate the stability of individual coagulation factors, clotting times, and other parameters of hemostasis in stored canine plasma. Methods Citrated plasma samples were obtained from 21 dogs. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and factor I, II, V, VII, VIII, IX, X, XI, and XII activities were measured on an automated coagulation analyzer with commercially available reagents. Antithrombin (AT) activity and D-dimer concentration were measured on an automated chemistry analyzer using validated kits. Samples were analyzed within 1 hour after collection (initial analysis) and once daily for 2 or 4 consecutive days following storage at room temperature (RT) or 4 degrees C, respectively. Results Storage time at either temperature did not have any effect on PT, factor II, V, VII, X, or XII activities, D-dimer concentration, or AT activity. In contrast, aPTT was significantly prolonged after 72 and 96 hours at 4 degrees C; fibrinogen concentration was decreased after 48 hours at RT; the activities of factors VIII and IX were decreased after 48, 72, and 96 hours at 4 degrees C; and factor XI activity was decreased after 72 hours at 4 degrees C. Conclusions Results suggest that storage of canine plasma for 2 days at RT does not have a significant effect on hemostasis test results with the exception of a slight decrease in fibrinogen concentration. In contrast, aPTT and factors VIII, IX, and XI were unstable in refrigerated plasma after 48 or 72 hours of storage.

A glico-hemoglobina (GHb) é um parâmetro importante no controle glicêmico de pacientes com DM. Vários estudos clínicos mostraram claramente que a melhora no controle glicêmico está fortemente associada com a diminuição no desenvolvimento e/ou progressão das complicações em diabetes melito tipos 1 e 2. A medida exata e precisa da GHb é uma questão importante para os laboratórios clínicos. Vários fatores afetam os resultados e podem levar a resultados errôneos. Nesta revisão, discutimos os problemas da padronização da determinação da GHb para monitorar a terapia diabética e também os principais fatores interferentes. Os métodos para GHb podem ser diferentemente afetados pelas interferências. O efeito da interferência pode ser clinicamente mais relevante com o pior controle glicêmico. O laboratório deve estar atento para estes fatores para evitar confusão na interpretação clínica dos resultados, e os clínicos devem contatar o laboratório sempre que houver discrepância entre a impressão clínica e o resultado laboratorial. Glycohemoglobin (GHb) has a key role in the assessment of glycemic control in diabetic patients. Several studies have clearly shown that improved glycemic control is strongly associated with decreased development and/or progression of diabetic complications in both type 1 and 2 diabetes mellitus. Therefore, accurate determination of GHb concentration is an important issue for clinical laboratories. Several factors may affect and lead to erroneous results. We discuss the problems of standardization of GHb measurements for monitoring glycemic control and also consider the potential interfering factors on GHb measurements. Moreover, GHb assays may be affected by interference in different ways. The effect of interference may be more clinically relevant with poor metabolic control. Laboratory staff must be aware of all pitfalls to avoid adding more confusion to the clinical interpretation of HbA1c values and physicians should contact laboratories if discrepancies between clinical impressions and laboratory data are observed.

The storage of soil samples for PLFA analysis can lead to shifts in the microbial community composition. We show here that conserving samples in RNAlater, which is already widely used to store samples for DNA and RNA analysis, proved to be as sufficient as freezing at −20 °C and preferable over storage at 4 °C for temperate mountain grassland soil. The total amount of extracted PLFAs was not changed by any storage treatment. Storage at 4 °C led to an alteration of seven out of thirty individual biomarkers, while freezing and storage in RNAlater caused changes in the amount of fungal biomarkers but had no effect on any other microbial group. We therefore suggest that RNAlater could be used to preserve soil samples for PLFA analysis when immediate extraction or freezing of samples is not possible, for example during sampling campaigns in remote areas or during transport and shipping., Highlights ► We tested storage of soils in RNAlater for analysis of phospholipid fatty acids. ► Storage in RNAlater proofed to be comparable to freezing at −20 °C. ► Freezing and RNAlater led to reduced fungal biomarkers compared to controls. ► Storage in RNAlater is suggested as a novel approach for sampling at remote areas.