Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

RNA preservation of Antarctic marine invertebrates

Fifteen species of marine invertebrate commonly occurring in the near-shore environment of Rothera base, Antarctica, were used to test tissue sample storage protocols with regard to preservation of RNA integrity. After animal collection, the tissues were either immediately extracted for RNA or stored at −80°C after having been, either directly flash frozen in liquid nitrogen or preserved in a commercial RNA storage solution, for extraction in the UK. In four cases, direct flash freezing produced enhanced RNA integrity compared with samples in the commercial storage solution. A subset of samples were further tested for the preferred temperature of storage in the commercial reagent. RNA integrity was well preserved at both +4 and −20°C over periods of 2 months, but degradation was rapid in tissues stored at room temperature. Eight out of the fifteen species only produced a single ribosomal band on gel electrophoresis. This survey provides a guide for tissue transport of Polar cold water marine invertebrates.     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

Long-term stability of oxidative stress biomarkers in human serum

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at ?20 and ?80?°C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at ?80?°C, but not in samples stored at ?20?°C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at ?80?°C. ROM and BAP can also be stored at ?20?°C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.     

Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Metabolic changes in human skin preserved at −3 and at −196 °C

Full-thickness human skin samples from living donors or split-thickness cadaver skin samples were stored at ?3 °C and at ?196 °C. In both circumstances, additions to the storage medium of cortisol, free l-amino acids, or a combination of both, resulted in an increased protection of the stored tissue. A most striking protective effect on protein synthesizing activity and on the membrane integrity of cutaneous cells was obtained by the addition of adenosine -5′-triphosphate (ATP) to the various storage media.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4°C, and more than a year at ?20°C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.     

野生鸡枞菌种长期保存方法比较

野生鸡枞菌种质资源的有效保存是对野生鸡枞加以保护和利用的前提。以自行分离的5个野生鸡枞菌株作为研究对象,采用蒸馏水保藏法和-80°C冻结保藏法对野生鸡枞菌种长期保存的方法进行了实验研究,蒸馏水法分别保存于室温和4°C,-80°C冻结保藏同时采用程控降温法和泡沫盒降温法,保存20个月后对4种不同方法保存的5个菌株的保存效果进行比较。实验结果表明:蒸馏水室温保存法菌种存活率为100%,萌发期较短,为4-10 d,是一种简便、实用、有效而成本低廉的长期保存方法;-80°C冻结保藏法的存活率为56%-76%,萌发期7-16 d,泡沫盒降温法可以很好地控制降温速度,是一种简便有效的控温方法。     

Stability of Solubilized Benzodiazepine Receptors

ABSTRACT

According to the observations of other researchers, benzodiazepine receptors solubilized with sodium deoxycholate are unstable, but stability can be improved by exchanging deoxycholate for Triton X-100. In our experiments we conclude that the choice of detergent is not the restrictive factor for the stability of the solubilized receptors, but the storage conditions are. Solubilized receptors, either stored in sodium deoxycholate or in Triton X-100, were stable for at least 2 months when stored at ?80°C, but both preparations were strongly unstable when stored at ?20°C. Alternatively, sodium deoxycholate-solubilized receptors may be lyophilized and then stored at ?20°C.     

Long-term stability of parameters of antioxidant status in human serum

Abstract

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, ? 20, ? 70 (or ? 80), and ? 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at ? 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at ? 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at ? 70/80°C is to be preferred for longer storage times.     

Assessment of protein stability in cerebrospinal fluid using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein profiling

Recent studies have evaluated proper acquisition and storage procedures for the use of serum or plasma for mass spectrometry (MS)-based proteomics. The present study examines the proteome stability of human cerebrospinal fluid (CSF) over time at 23°C (room temperature) and 4°C using surface-enhanced laser desorption/ionization time-of-flight MS. Data analysis revealed that statistically significant differences in protein profiles are apparent within 4 h at 23°C and between 6 and 8 h at 4°C. Inclusion of protease and phosphatase inhibitor cocktails into the CSF samples failed to significantly reduce proteome alterations over time. We conclude that MS-based proteomic analysis of CSF requires careful assessment of sample collection procedures for rapid and optimal sample acquisition and storage.     

A simple method for cryopreservation of MDBK cells using trehalose and storage at −80°C

Madin Darby bovine kidney cells were stored at ?80°C using trehalose. Trehalose was loaded into the cells by fluid-phase endocytosis that was facilitated by heat shock at 40°C for 1 h. Loaded cells were gradually frozen and stored at ?80°C. Revival of cells was done by quick thawing and immediately seeded in the tissue culture flasks. The membrane integrity of cells was measured at different times post-storage by trypan blue dye exclusion method. It was estimated to be 96.23, 73.84, 57.33, 54.36, 25.47, 50.53 and 46.86% at 0, 7, 60, 90, 120, 160 and 180-day post-storage, respectively. Cryostorage of cells at ?80°C may help to reduce the use of liquid nitrogen.     

Effects of Long‐Term Storage Methods on the Infectivity of Urediospores of Phakopsora pachyrhizi

A method for long‐term storage of spores of Phakopsora pachyrhizi was optimized. Three methods with different procedures for spore harvest and four different reactivation methods (varying in hydration or using heat shock) were analysed for the suitability for long‐term storage at ?80°C. All conservation methods as well as all reactivation methods lead to the infection of soybean leaves after 1 year of storage. Regarding efficiency and labour input, the most recommended method is to tap off spores from infected and sporulating leaves with subsequent dehydration before storage at ?80°C. Because hydration or heat shock steps did not provide any advantages, spores can be suspended in Tween water directly after storage and used as inoculum.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4?°C for up to 7 days. We also analyzed storage at ?80?°C across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24?h, followed by shipping the samples on ice over 2–3?days, and finally, long-term storage in the ?20?°C or ?80?°C freezer.     

Effect of temperature and ripening stages on membrane integrity of fresh-cut tomatoes

The shelf-life of fresh-cut tomatoes mainly depends on loss of tissue integrity and firmness that occurs also in intact fruits after long-term cold storage due to chilling injury. Round-fruit tomatoes (Solanum lycopersicum L.) cv. Jama were stored in 1.1-L plastic (polyethylene) fresh-cut produce containers as 10.0-mm-thick tomato slices and as intact tomatoes at 4 ± 0.5 °C. The aim of this work was to study the loss of membrane integrity and biochemical processes involved in membrane disruption. Electrolyte leakage and lipid peroxidation were studied at different stages of maturity: mature green, pink (PK), fully ripe and two different storage temperatures: 4 and 15 °C. The tomato slices of PK stage stored at 4 °C did not show changes for both parameters, while significant increase in membrane leakage and lipid peroxidation was observed at 15 °C, especially after 24 h of storage. The enzymes showed a simultaneous increase in their activities with a rise in electrolyte leakage and lipid peroxidation after 7 days of storage. Finally, phospholipase C (PLC) and phospholipase D (PLD) were investigated for intact fruit and tomato slices stored at 4 °C. The PLC had higher activity compared with PLD. In conclusion, the loss of membrane integrity in fresh-cut tomatoes is mainly affected by ripening stages, storage temperature and duration. The wounds enhance the PLC and PLD activities and they play a role late during storage.