Storage Time Points Research Articles

Elevated levels of thrombin‐generating microparticles in stored red blood cells

During storage, red blood cells (RBCs) lose their membrane stability, leading to haemolysis and microparticle (MP) formation. The use of RBCs stored for more than 28days has been associated with an increased incidence of deep vein thrombosis. However, the exact mechanism by which coagulation activation is enhanced in stored RBCs is still unknown. To investigate the relevant potential procoagulant activities of MPs and study the relative procoagulant factors for initiating the coagulation on MPs in stored RBCs. MPs were isolated from the plasma of RBC units stored in citrate-phosphate-dextrose-adenine. At seven storage time-points (d0, d7, d14, d21, d28, d35 and d42), MPs were morphologically observed, quantified and analysed for tissue factor, factor XI (FXI) and their thrombin-generating potential. MPs were observed using electron microscopy. The size of the MPs ranged from 0·272μm to 0·973μm in diameter. During the storage of RBCs in plastic bags, the MP concentration increased from 3389±218/μl at day 0 to 61586±2237/μl at d42. Thrombin generation was dependent on the total number of MPs (r=0·987). Anti-human FXI antibody inhibited thrombin concentrations by 50·3% compared with control plasma, whereas antitissue factor and antitissue factor pathway inhibitor failed to reduce thrombin concentrations. Our study provides evidence that MP formation due to RBC storage might propagate coagulation not only by exposing phosphatidylserine, but also by initiating thrombin generation independently of tissue factor in a FXI -dependent manner.

Factors affecting oxidation stability of commercially available biodiesel products

Factors affecting oxidation stability for several commercially available biodiesels were primarily investigated by acid value (AV) and induction period (IP) evaluations in this study. It was found that the measured IP at different storage time points were somewhat dependent on the saturated degree of fatty acid methyl esters (FAMEs), the corresponding measured AV scattered randomly. Generally, AV increased and IP decreased after one year of storing in a dark cold room in an air‐tight tank. Solvents (methanol, acetone and water) did not show a contribution to altering IP. Metals (copper and lead) showed the strongest detrimental effects to oxidative stability although somewhat depending on the particle size and oxide coating thickness, however, aluminum alloy and steel were not the case. Among the antioxidants, pyrogallol (PY) was the best in enhancing IP with a concentration of less than 3000ppm, however, tert-butylhydroquinone (TBHQ) was the best after 3000ppm, followed by propyl gallate (PG), butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxyltoluene (BHT), and α-tocopherol. The appropriate dosage of PY was also evaluated to achieve the specified IP regulated by EN-14112 for samples with copper or lead contamination.

Plant Sterols and Antioxidant Parameters in Enriched Beverages: Storage Stability

Plant sterols (PS) stability, antioxidant parameters, and color were studied during 6 months of storage at 4, 24, and 37 °C in three PS-enriched functional beverages. Beverages were skimmed milk with fruit juice and PS (MFJPS), fruit juice and PS (FJPS), and skimmed milk with PS (MPS). No loss in total PS content occurred during storage observing the same values at any given storage time point. Total carotenoids decreased 36% with storage at two months and then remained stable. Total polyphenols showed fluctuations throughout the storage, remaining stable at 6 months and reaching initial values. The antioxidant capacity (TEAC method) increased 18% at 6 months, and there was an increase in color over time and temperature, probably due to Maillard reaction compound formation. The increase in total antioxidant capacity might have helped PS maintenance throughout storage, these beverages being a good PS source even after 6 months of storage.

Effects on 3-Mercaptohexan-1-ol Precursor Concentrations from Prolonged Storage of Sauvignon Blanc Grapes Prior to Crushing and Pressing

Formation of wine thiol precursors is a dynamic process, which can be influenced by vineyard and winery processing operations. With the aim of increasing thiol precursor concentrations, a study of the effects of storing machine-harvested Sauvignon blanc grapes prior to crushing and pressing was undertaken on a commercial scale. 3-Mercaptohexan-1-ol (3-MH) precursors, 2-S-glutathionylcaftaric acid (grape reaction product, GRP), glutathione (GSH) and a number of C6 compounds were assessed at several time points during the experiment. The concentration of the cysteine precursor to 3-MH doubled within 8 h and tripled after 30 h while the GSH and cysteinylglycine precursors increased in concentration roughly 1.5 times. (E)-2-Hexenal and GSH levels decreased as thiol precursors, GRP and C6 alcohols increased during storage. Principal component analysis revealed that precursors contributed to most of the variation within the samples over the storage period, with additional influence, primarily from GSH and GRP, as well as (E)-2-hexenal and (Z)-3-hexen-1-ol. Early storage time points were associated with higher concentrations of GSH and some unsaturated C6 compounds while longer storage times were most closely associated with higher thiol precursor and GRP concentrations. This study provides a detailed overview of interactions related to thiol precursor formation on a commercial scale and highlights the ability to manipulate precursor concentrations prior to grape crushing.

Quantitative and qualitative analysis of Botrytis inoculated on table grapes by qPCR and antibodies

The fungus Botrytis cinerea is the major cause of decay in table grapes during storage, and the severity of decay depends in part on contamination with the fungus before storage. The current SO 2 technology to prevent decay is robust and independent of the level of contamination by B. cinerea. The introduction of alternative technologies may however require implementation of means which are proportional to the level of contamination. The objectives of this study were to test the feasibility of quantifying B. cinerea in artificially inoculated grapes and to monitor the progress of disease during storage. Two methods were compared for detection of B. cinerea in grapes; an antibody kit specific for B. cinerea, and quantitative PCR using fungal specific primers. Antibodies for fast detection of B. cinerea yielded positive results only in the later stages of decay development. In contrast, the quantitative PCR demonstrated positive identification of the fungus at all storage time points, and found increasing amounts of the fungus during storage.

Long‐term release of monomers from modern dental‐composite materials

The elution of monomers from composite materials influences the biocompatibility of dental restorations. The purpose of the present study was to investigate the elution of monomers [bisphenol A glycidyl methacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A (BPA)] from two light-cured materials (nanohybrid and ormocer) and from a chemically cured composite material, after different curing times (0, 20, 40 and 80 s) and different storage periods (24 h, 7 d, 28 d, and 1 yr after curing). Each specimen was stored in 1 ml of 75% ethanol. This medium was renewed after 24 h, 7 d, 28 d, and 1 yr. The ethanol samples were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The amount of monomers released from the nanohybrid and the chemically cured composite was significantly higher than released from the ormocer. The curing time exerted a significant effect on the release of monomers. For the nanohybrid, less monomer was released after increasing the curing time. For the ormocer, 80 s of curing resulted in a higher degree of monomer release. The effect of storage differed between the monomers. Although the elution of TEGDMA was significantly decreased after storage for 28 d and 1 yr, a similar amount of BisGMA was released at each storage time-point analyzed, even after 1 yr. The present study showed that ormocer released a very small amount of monomers compared with the other materials.

Evaluation of the functionality of cooled stored sperm with three commercial extenders

s / Theriogenology 70 (2008) 1384–1404 1393 and parameters of sperm motility. Furthermore, analysis of data according to the total sperm number per dose revealed a reduction of sperm motility with the decreasing number of spermatozoa. Doses with 1.1 10 cells had a lower percentage of motile spermatozoa (SpermVision) after 72 h of storage than doses with 2.2 10 cells. In doses of 0.6 10 cells, a significant drop was already detectable after 24 h. A thermoresistance test (incubation for 300 min at 38 8C) with 72-h old semen samples containing 0.6 10 cells revealed a lower motility (P 70% motility and normal acrosome at 2 d preservation of liquid semen at 4 8C were defined as cold shock resistant (Boar A), whereas sperm with 75%, and rate of affected acrosomes was <20%. Over all storage time points, sperm had decreasing absolute values of motility parameters in bicarbonate-free medium. Nevertheless, sperm motility still could be stimulated by bicarbonate in terms of specific kinematic parameters (e.g. path velocities).

Comparative Proteome Analysis To Study Novel Markers of the Blood Platelet Lesion.

Platelet transfusion is a very common live-saving medical procedure for patients with platelet-deficient diseases like leukemia. In contrast to other blood components, the availability of platelets is restricted since they have a limited shelf-life of 5 days for transfusion purposes. This is due to storage-related deterioration in product quality resulting in the clearance from circulation. To overcome this problem, it is important to understand the molecular mechanisms leading to blood platelet lesion during storage. We were using two different proteomic approaches combined with functional biochemistry to investigate time-dependent changes in the blood platelet proteome. One type of analysis consisted of the separation of the platelet proteome at two different time points of storage, day 1 and day 8, using 2-dimensional (2D) gel electrophoresis for qualitative and DIGE technology for quantitative analysis. The identification of proteins changing in spot intensity was carried out by liquid chromatography and tandem mass spectrometry. The second method was based on stable isotope labeling with ITRAQ/ICAT reagents in combination with protease treatment, equivalent mixing, separation of the resulting peptides and quantitative analysis by mass spectrometry. Taken together, for the 2D/DIGE approach we analyzed 977 spots corresponding to 103 different proteins and for the ITRAQ approach 1428 peptides corresponding to 355 proteins, resulting in 37 proteins significantly changing both quantitatively due to protein synthesis or degradation and qualitatively due to post-translational modification and enzymatic activity. The high degree of correlation between the two approaches validates the experimental set-up and confirmed the requirement for complementary tools to enhance proteome coverage. Among others, increased amounts of integrins and other proteins known to form receptor signaling complexes with these integrins as well as proteins observed in platelet activation were detected. This proves, for the first time, that there is an apparent link between blood platelet storage lesion and cell signaling.

Study of Precursors Responsible for Off-Flavor Formation during Storage of Potato Flakes

Off-flavors frequently appear during the storage of potato flakes. Volatile profile analysis performed by solid-phase microextraction-gas chromatography-mass spectrometry revealed that hexanal is the main compound that appears during the storage period. Hexanal may be a degradation product of linoleic acid formed through linoleic acid hydroperoxide cleavage. Profiles of hexanal precursors were determined from potato flakes at different storage time points. Linoleic acid-derived oxylipins are predominant in potato flakes. The free oxylipins identified, in descending order, are as follows: hexanal, hydroxy polyunsaturated fatty (PUFAs), oxo PUFAs, divinyl ether PUFAs, and hydroperoxy PUFAs. However, the main oxylipins detected were esterified: esterified hydroxy, hydroperoxy, and oxo PUFAs. Oxylipins reveal different evolutions during the storage period. Chiral high-performance liquid chromatography analysis of the precursors of hexanal and other oxylipins revealed a racemic composition that supports the nonenzymatic formation of hexanal and most of the other oxylipins identified.

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6–16 h. Gene expression by real-time PCR of cfos, HIF1α, Bcl2, PCNA, TGFβ1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2–3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

Leucocyte-Reduced Buffy Coat Platelet Concentrates (BCPC) Treated with INTERCEPT™ Stored up to 7 Days: Hemostatic Function in a Whole Blood Flow System.

Background: The risk of bacterial growth has limited the shelf life of platelet concentrates (PC) to 5 days. Modern platelet storage containers facilitate storage for up to 7 days, if bacterial contamination is prevented. INTERCEPT (Baxter, La Chatre, France; Cerus, Concord, CA) photochemical treatment (PCT) for pathogen reduction based on amotosalen (150μM) and UVA illumination (3 J/cm2) inactivates high titers of bacteria in PC (Transfusion 2004; 44: 1496–1504). Adhesion and aggregation of platelets to injured vascular surfaces are critical aspects of platelet hemostatic function. In this study, the adhesion and aggregation of leucocyte-reduced buffy coat derived PC (BCPC), treated with INTERCEPT and stored up to 7 days, were measured on injured vascular surfaces using an ex-vivo blood flow system.Methods: BCPC were prepared from 450 mL-whole blood donations with the top and bottom method (Optipress II, Baxter). Five BCPC, of the same ABO group, were pooled with additive solution (Intersol™) the day following collection, after viral screening testing was completed. Following centrifugation and leukocyte depletion, two BCPC pools of the same ABO group were mixed and divided. One pooled BCPC was treated with INTERCEPT (I-BCPC) and the other was prepared by conventional methods (C-BCPC); and both were stored in 1.3 liter PL2410 plastic containers (Baxter R4R7012) at 22 ± 2°C with continuous agitation for 7 days. Samples for hemostatic function testing were taken immediately after preparation before splitting for treatment and after 5 and 7 days of storage. Platelet counts were performed in K3EDTA in a Coulter MD II counter (Coulter, Miami, FL). Samples of I-BCPC and C-BCPC were added to citrate anticoagulated blood, previously depleted of platelets and leukocytes by filtration, and adjusted to a final platelet count of 150x109/L. Enzymatically denuded vascular segments were exposed to circulating whole blood, reconstituted with I-BCPC and C-BCPC, in Baumgartner chambers at a shear rate of 800 s−1 for 10 minutes. The proportion (%) of the vascular surface area covered by platelets after perfusion was measured for each type of BCPC (N = 9) at each storage time point. Platelets and groups of platelets were classified as adhesive when platelet masses were less than 5 μm in height and as thrombi when height exceeded 5 μm. Data were analyzed with the SPSS 12.0.1 statistical package with significance at p < 0.05, and expressed at the mean ± SEM Results(Table).Conclusion: The platelet count of I- BCPC decreased by 12.3% including PCT processing losses and 7 days of storage. However, I- BCPC platelet adhesive and aggregatory capacities under flow conditions were similar to C- BCPC, and were well conserved for up to 7 days of storage.Hemostatic Function of Stored I-BCPC and C-BCPCParameterI-BCPCC-BCPCpDay 1(Pre Treatment)Platelet Count (109/L)945±40945±40Platelet Coverage (%)26.0±3.726.0±4.2Adhesion(%)24.0±3.724.0±3.7Thrombus(%)1.9±0.61.9±0.6Day 5 StoragePlatelet Count (109/L844±41902±440.004Platelet Coverage (%)20.9±2.220.6±1.60.9Adhesion(%)19.9±2.119.3±1.40.8Thrombus(%)0.9±0.31.2±0.40.5Day 7 StoragePlatelet Count (109/L)829±32923±480.008Platelet Coverage (%)27.1±2.921.2±2.80.06Adhesion(%)26.0±2.720.4±2.70.06Thrombus(%)1.2±0.30.7±0.20.16

Bioactive Compounds of Grapefruit (Citrus paradisiCv. Rio Red) Respond Differently to Postharvest Irradiation, Storage, and Freeze Drying

In the present study, the effect of irradiation, storage, and freeze drying on grapefruit bioactive compounds was investigated. Grapefruits were exposed to one of two irradiation doses: 0 (control) or 300 Gy (137Cs, a proposed treatment against fruit flies) and then stored for up to 6 days. At the last storage time point (6 days after harvest), grapefruit pulp from control and irradiated fruits was freeze-dried. Bioactive compounds were extracted from Rio Red grapefruit pulp and analyzed with reverse phase liquid chromatography while volatile compounds were analyzed using gas chromatography. Freeze-dried pulp from irradiated fruits had a higher (P < or = 0.05) flavonoid content (naringin and narirutin) as compared to the freeze-dried pulp from the control fruits. Freeze-drying treatment reduced (P < or = 0.05) the lycopene content, but the reduction (P < or = 0.05) in beta-carotene content occurred only in the control fruit. Reduction in d-limonene and myrcene was observed in the irradiated fruits at 6 days after harvest and in the freeze-dried samples. These results warrant testing of the effect of postharvest treatments and processing on bioactive compounds in functional systems as they have varied effects on different bioactive compounds of grapefruit.

Assessment of complement activation during membrane‐based plasmapheresis procedures

Previous studies have suggested that plasmapheresis procedures using a separation membrane may activate the complement system and release anaphylatoxins. This study determines the content in C3a/C3a(des Arg) and C5a/C5a(des Arg) in plasma donations obtained by the new Haemonetics Filter Core (FC) procedure and compares it to Baxter Autopheresis C (Auto-C). FC performs sequential blood centrifugation and plasma filtration on a microporous polyethersulfone membrane, while Auto-C removes blood cells by simultaneous gravitation and filtration on a rotating nylon membrane. One group of 34 donors donated on FC and two groups of 30 and 10 donors on Auto-C. Plasma aliquots were taken from the plasma units within 30 min of the end of the collection procedures, frozen at < -30 degrees C and assessed for C3a and C5a at various time points of storage. Mean C3a/C3a(des Arg) in FC plasma (N = 34) was 1,151 (range: 526-2,991), 1,092 (range: 349-3498), and 507 (range: 307-815) ng/ml at time of collection and after 6 and 12 months of storage, respectively. Respective C5a/C5a(des Arg) was 26.6 (range 4.9-74), 18.9 (9.5-42.6), and 30.9 (range: 10.7-62.3) ng/ml. Mean C3a/C3a(des Arg) was higher in Auto-C (P < 0.001): 4,724 ng/ml (N = 10; range: 2,400-7 ,360) and > 4,149 ng/ml (N = 30; 2,408- > 6,430) after 3 and 18 months storage, respectively. Mean C5a/C5a(des Arg) was 32.1 ng/ml (N = 30; range: 10.6-57.2) after 18 months of storage. Complement activation in FC plasmas appears limited compared to Auto-C, suggesting better biocompatibility of this collection device and/or a favourable impact of the sequential cell centrifugation/filtration technology used. Further studies are needed to explain differences in complement activation between apheresis procedures and to assess clinical impacts, if any.

Stability study of fotemustine in PVC infusion bags and sets under various conditions using a stability-indicating high-performance liquid chromatographic assay

The stability and compatibility of fotemustine, a nitrosourea anticancer agent, in 5% dextrose solution with polyvinyl chloride (PVC) containers and administration sets were studied under different conditions of temperature and light. The drug was diluted to 0.8 and 2 mg ml −1 in 100 or 250 ml 5% dextrose injection solutions for 1-h simulated infusions using PVC bags and administration sets with protection from light. After preparation in the PVC bags containing 5% dextrose, fotemustine was also prepared at the same concentrations and stored at 4°C for 48 h and at room temperature (22°C) or at sunray exposure (>30°C) over 8 h with or without protection from light. The solution samples were removed immediately at various time points of simulated infusions and storage, and stored at −20°C until analysis. The physical compatibility with PVC and chemical stability in solution of fotemustine were assessed by visual examination and by measuring the concentration of the drug in duplicate using a stability-indicating high-performance chromatographic assay. When admixed with a 5% dextrose solution, fotemustine 2 and 0.8 mg ml −1 was compatible and stable over 1-h of simulated infusion using PVC bags through PVC administration sets with protection from light. On the other hand, in the same diluent, fotemustine was compatible and stable with PVC bags for at least 8 h at 22°C with protection from light and for at least 48 h at 4°C with protection from light. There were no pH variation, no visual change, no color change, no visible precipitation and no loss of the drug. Conversely, when the solutions were exposed to light (ambient or solar), the drug concentration decreased rapidly, leading to the production of a degradation product as shown by mass spectral analysis and a discoloration of the solutions. Finally, in all cases, no DEHP (di-2-ethylhexyl phthalate) was detected in the injection solution.