Stability of antioxidant vitamins in whole human blood during overnight storage at 4°C and frozen storage up to 6 months

2018 ◽  
Vol 88 (3-4) ◽  
pp. 151-157 ◽  
Author(s):  
Scott W. Leonard ◽  
Gerd Bobe ◽  
Maret G. Traber
Keyword(s):  
Ascorbic Acid ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Frozen Storage ◽  
Blood Collection ◽  
Plasma Samples ◽  
Optimal Conditions ◽  
Plasma Ascorbic Acid ◽  
Blood Samples ◽  
Sample Handling

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

scholarly journals P1611PRE-ANALYTICAL CONSIDERATIONS IN STUDYING CIRCULATING MICRORNA EXPRESSION: COMPARISON BETWEEN PAIRED EDTA PLASMA, EDTA WHOLE BLOOD AND PAXGENE BLOOD RNA TUBES

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Veerle Wijtvliet ◽  
Amaryllis Van Craenenbroeck ◽  
Annemieke Smet ◽  
Steven Van Laere ◽  
Annick Massart ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Whole Blood ◽  
Blood Collection ◽  
Circulating Microrna ◽  
Plasma Samples ◽  
Circulating Mirnas ◽  
Blood Samples ◽  
Edta Plasma ◽  
Whole Blood Samples ◽  
Count Table

Abstract Background and Aims microRNA (miRNA) dysregulations have been related to pathological processes, including kidney disease. Relative stability in blood makes miRNAs attractive biomarkers. The current recommendation is to use fresh EDTA plasma samples (i.e. processed within 30 min. from sampling) to study circulating miRNA. However, cumbersome logistics might preclude broad implementation. Therefore, we investigated the potential of whole blood EDTA and PAXgene blood RNA tubes as alternative sources to study circulating microRNA expression profiling. Method Paired EDTA plasma, EDTA whole blood and PAXgene blood RNA tubes were obtained from 10 healthy adults (50% male). EDTA plasma samples were processed within 30 min. after sampling and immediately stored at -80°C. EDTA whole blood tubes and PAXgene tubes were kept at room temperature for 48 hours after sampling. Subsequently, the content of the EDTA whole blood samples was transferred to a 15 mL Falcon tube and stored at -80°C. PAXgene tubes were transferred to -20°C following the manufacturer’s protocol. Within 1 month of storage, all samples were thawed and miRNA was extracted using the Qiagen miRNeasy serum/plasma kit and subjected to RNA-sequencing (Oxford Genomics Centre). Based on the raw data, a count table was created using the online tool miRDeep* for the identification of both novel and known microRNAs. Subsequent downstream bio-informatic analyses approaches consisted of 1) unsupervised hierarchical clustering with principal component analysis (PCA); 2) calculation of differential miRNA expression using generalized linear models with differences considered significant if the false discovery rate-adjusted p-value was inferior to 10%. Results Initial assessment of the count table showed significant differences in the number of detected microRNAs. A median of 220 different microRNAs was detected in EDTA plasma samples versus 661 in PaxGene samples (p < 0.05) and 490 in EDTA whole blood samples (p < 0.05) (Figure 1A). We also found fewer novel miRNAs in EDTA plasma samples than in PAXgene samples (p < 0.001) and EDTA whole blood samples (p < 0.05). Low count microRNAs, defined as below 10 reads in more than 20% of the samples, were more abundant in Paxgene samples versus EDTA plasma samples (p = 0.0039), but this difference was not significant when comparing EDTA whole blood samples with EDTA plasma samples (Figure 1B). PCA analysis (Figure 1C) showed a clear separation of samples according to the blood collection method, strongly suggesting that the blood collection method predominantly determines the miRNA expression profile. Conclusion Bio-informatic analyses demonstrated different miRNA expression profiles according to three different blood collection methods, underpinning the importance of a standardized method for the collection of blood aimed at studying circulating miRNAs. As such, this study has important implications for the design of novel studies aiming to investigate circulating miRNAs.

Search for Melanoma Markers in Plasma and Serum Samples

2005 ◽  
Vol 11 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Roberta Seraglia ◽  
Susanna Vogliardi ◽  
Graziella Allegri ◽  
Stefano Comai ◽  
Mario Lise ◽  
...  
Keyword(s):  
High Frequency ◽  
Healthy Subjects ◽  
Ionic Species ◽  
Low Molecular Weight ◽  
Ionization Mass ◽  
Plasma Samples ◽  
Serum Samples ◽  
Blood Samples ◽  
Melanoma Patients ◽  
Diagnostic Ions

Fourteen blood samples from patients with melanomas and 11 blood samples from healthy subjects were analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The study focussed on species of low molecular weight, in the 800–5000 Da range, present in plasma and sera. While for healthy subjects plasma samples lead to the production of a higher number of ionic species, for melanoma patients a high number of diagnostic ions, present with high frequency and with quite high relative abundance, are present, in particular, in serum samples and, to a lesser extent, also in plasma. Since plasma samples are obtained more easily in comparison to sera, it is possible to suggest that plasma can also be used for these studies.

scholarly journals Determination of Total Interleukin-8 in Whole Blood after Cell Lysis

2000 ◽  
Vol 46 (9) ◽  
pp. 1387-1394 ◽  
Author(s):  
Jochen Reinsberg ◽  
Jörg Dembinski ◽  
Christoph Dorn ◽  
Daniela Behrendt ◽  
Peter Bartmann ◽  
...  
Keyword(s):  
Whole Blood ◽  
Healthy Subjects ◽  
Room Temperature ◽  
Sample Pretreatment ◽  
Cell Lysis ◽  
Interleukin 8 ◽  
Simple Method ◽  
Sample Handling ◽  
A Cell

Abstract Background: It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects. Methods: Total IL-8 content of whole blood was determined after lysing blood cells with Milenia® cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE® IL-8 immunoassay. Results: When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 ± 13 ng/L to 114 ± 16 ng/L (n = 10). In lysate stored for 48 h at 4 °C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% ± 5% (n = 12) of initial concentration was recovered. From lysate frozen at −20 and −80 °C, respectively, 84% ± 4% and 93% ± 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3.2–4.2%) and blood lysate (CV, 3.7–4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% ± 3%) was observed; for lysate specimens with a cell lysis solution content ≥75%, the recovery after dilution was 98% ± 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% ± 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22–61 years) was 83 ng/L of blood (2.5–97.5 percentile range, 49–202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome. Conclusions: The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.

scholarly journals Quality Control of Serum and Plasma by Quantification of (4E,14Z)-Sphingadienine-C18-1-Phosphate Uncovers Common Preanalytical Errors During Handling of Whole Blood

2018 ◽  
Vol 64 (5) ◽  
pp. 810-819 ◽  
Author(s):  
Xinyu Liu ◽  
Miriam Hoene ◽  
Peiyuan Yin ◽  
Louise Fritsche ◽  
Peter Plomgaard ◽  
...  
Keyword(s):  
Whole Blood ◽  
Plasma Samples ◽  
Blood Samples ◽  
Disease States ◽  
Long Term Storage ◽  
Reliable Assessment ◽  
Therapeutic Decisions ◽  
Life Threatening ◽  
Research Findings

Abstract BACKGROUND Nonadherence to standard operating procedures (SOPs) during handling and processing of whole blood is one of the most frequent causes affecting the quality of serum and plasma. Yet, the quality of blood samples is of the utmost importance for reliable, conclusive research findings, valid diagnostics, and appropriate therapeutic decisions. METHODS UHPLC-MS-driven nontargeted metabolomics was applied to identify biomarkers that reflected time to processing of blood samples, and a targeted UHPLC-MS analysis was used to quantify and validate these biomarkers. RESULTS We found that (4E,14Z)-sphingadienine-C18-1-phosphate (S1P-d18:2) was suitable for the reliable assessment of the pronounced changes in the quality of serum and plasma caused by errors in the phase between collection and centrifugation of whole blood samples. We rigorously validated S1P-d18:2, which included the use of practicality tests on >1400 randomly selected serum and plasma samples that were originally collected during single- and multicenter trials and then stored in 11 biobanks in 3 countries. Neither life-threatening disease states nor strenuous metabolic challenges (i.e., high-intensity exercise) affected the concentration of S1P-d18:2. Cutoff values for sample assessment were defined (plasma, ≤0.085 μg/mL; serum, ≤0.154 μg/mL). CONCLUSIONS Unbiased valid monitoring to check for adherence to SOP-dictated time for processing to plasma or serum and/or time to storage of whole blood at 4 °C is now feasible. This novel quality assessment step could enable scientists to uncover common preanalytical errors, allowing for identification of serum and plasma samples that should be excluded from certain investigations. It should also allow control of samples before long-term storage in biobanks.

Coagulation Factor Activity in Therapeutic Plasma Prepared from Whole Blood with Pathogen Inactivation (INTERCEPT™) Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4149-4149
Author(s):  
Jean-Pierre Cazenave ◽  
Hervé Isola ◽  
Marie-Louise Wiesel ◽  
Daniel Kientz ◽  
Michel Laforêt ◽  
...  
Keyword(s):  
Whole Blood ◽  
Total Protein ◽  
Frozen Storage ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Blood Collection ◽  
Pathogen Inactivation ◽  
Factor Activity ◽  
Illumination Time ◽  
Frozen Plasma

Abstract Background. A photochemical treatment (PCT) using amotosalen HCl (S-59) and UVA light was developed to inactivate pathogens and leukocytes in therapeutic plasma (INTERCEPT™, I-FFP) frozen within 8 hr of collection. Previous studies demonstrated a broad spectrum of pathogen inactivation (Transfusion2006;46:1168) and clinical efficacy of I-FFP for support of coagulopathies (Transfusion2005;45:1362; Blood2006; 107:3753), and plasma exchange of TTP (Transfusion 2006;46). Preparation of therapeutic plasma from whole blood would complement blood center logistics and reduce the cost of therapeutic frozen plasma provided sufficient coagulation factors were retained. Aims. We measured coagulation factors in plasma isolated from whole blood held overnight at controlled temperature (21 ± 3°C), processed with pathogen inactivation, and frozen within 18 hr of blood collection. Methods. Whole blood units, approximately 460 mL, anticoagulated with CPD (Baxter, La Chatre, France) were drawn from group A, O, B and AB donors. Units were processed after 16 hr storage, and plasma was isolated by centrifugation. Two to 3 plasma units of matched blood group were pooled (n = 30: A = 14, O = 14, B = 1, AB =1) to a final volume of 635 mL. Baseline samples for assay of coagulation factors were withdrawn. Each of 30 pools was mixed with 15 mL of 6 mM amotosalen (150 uM: final concentration) and illuminated with a 3 J/cm2 UVA treatment. Following illumination (~ 8 min) and passage through a flow compound adsorption device (~20 min) to reduce levels of residual S-59, treated plasma units (650 mL) were divided into 3 equal storage units of ≥ 200 mL. Before freezing, post-treatment samples for assay of coagulation factors were withdrawn for assay of coagulation factors. Treated plasma units were flash frozen at -80°C, and transferred to −30°C for 12-month storage. Treated units were withdrawn after 1 month to measure total protein, albumin, IgG, IgM, IgA, fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, VIII-vWF, Proteins C and S, AT III, plasminogen, alpha-2 antiplasmin, D-dimers, PT, and APTT. Results. Baseline coagulation factor levels (Mean ± SD) were in suitable therapeutic ranges. After PCT, all units had residual platelets < 1×109/L, WBC < 1×104/L, and RBC < 1 × 109/L. After PCT and frozen storage for 1 month, total protein (59 ± 2 g/L), albumin (38 ± 1 g/L), IgG (8.9 ± 1.1g/L), IgA (1.8 ± 0.4 g/L) and IgM (0.9 ± 0.3 g/L) were unchanged from baseline. Mean values for fibrinogen (g/L), coagulation factors (IU/dL), coagulation inhibitors (IU/dL), were variably reduced from baseline, but within ranges defined as suitable for therapeutic plasma (Table). There was no evidence of plasma activation. Conclusions. Plasma prepared from whole blood after storage on cooling plates before processing with the INTERCEPT system for pathogen inactivation retained coagulation factor activity levels after frozen storage (−30°C) in conformance with French national standards for therapeutic frozen plasma (FP). Approximately 36 units (200 mL) could be prepared per hr of illumination time with this system.

scholarly journals Effects of storage temperature and repeated freeze–thaw cycles on stability of bovine plasma concentrations of haptoglobin and ceruloplasmin

2017 ◽  
Vol 29 (5) ◽  
pp. 738-740 ◽  
Author(s):  
Paulo G. M. A. Martins ◽  
Philipe Moriel ◽  
John D. Arthington
Keyword(s):  
Whole Blood ◽  
Storage Temperature ◽  
Plasma Concentrations ◽  
Freezing Temperature ◽  
Plasma Samples ◽  
Blood Samples ◽  
Freeze Thaw ◽  
Bovine Plasma ◽  
Whole Blood Samples ◽  
Freezing Temperatures

We evaluated the effects of storage temperature (−20 or −80°C) and handling procedure on plasma concentrations of bovine haptoglobin and ceruloplasmin. Within each temperature, whole blood samples were: centrifuged within 2 h of collection and plasma kept frozen until analysis (control); refrigerated at 4°C for 24 h before plasma harvest and freezing (24H); or plasma harvested and frozen within 2 h after collection, but then plasma samples were thawed and refrozen 1 wk (1X), 1 and 2 wk (2X), or 1, 2 and 3 wk (3X) before analyses. Haptoglobin concentrations were greatest at 24H, but similar among remaining treatments. Ceruloplasmin concentrations were not affected by the handling procedures. Storage temperature did not affect haptoglobin concentrations, but ceruloplasmin concentrations decreased when stored at −20 versus −80°C. Except for greater concentrations after 24 h storage at 4°C, haptoglobin concentrations remained stable at either freezing temperature and through freeze–thaw cycles. Ceruloplasmin concentrations decreased after 3 freeze–thaw cycles and required lower freezing temperatures to remain stable.

scholarly journals Effects of Storage and Type of Blood Collection Tubes on Hepatitis C Virus Level in Whole Blood Samples

2001 ◽  
Vol 39 (5) ◽  
pp. 1788-1790 ◽  
Author(s):  
H. H. Kessler ◽  
E. Stelzl ◽  
R. B. Raggam ◽  
J. Haas ◽  
F. Kirchmeir ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Whole Blood ◽  
Blood Collection ◽  
Blood Samples ◽  
Virus Level ◽  
Whole Blood Samples ◽  
Blood Collection Tubes

scholarly journals Quantitative Reverse Transcription-PCR Measurement of Thyroglobulin mRNA in Peripheral Blood of Healthy Subjects

1999 ◽  
Vol 45 (6) ◽  
pp. 785-789 ◽  
Author(s):  
Susan T Wingo ◽  
Matthew D Ringel ◽  
Jeffrey S Anderson ◽  
Aneeta D Patel ◽  
Yvonne D Lukes ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Reverse Transcription ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Thyroid Disease ◽  
Healthy Subjects ◽  
Extraction Technique ◽  
Blood Collection ◽  
Rt Pcr ◽  
Total Rna

Abstract Background: Thyroglobulin mRNA can be detected qualitatively in the peripheral blood of patients with metastatic thyroid cancer, thyroid cancer patients with residual thyroid bed uptake, and individuals with no known thyroid disease with intact thyroid glands by use of a lengthy, highly sensitive extraction technique. To improve and broaden the clinical usefulness of this assay, we developed a quantitative reverse transcription (RT)-PCR assay for thyroglobulin mRNA, using RNA recovered from whole blood with a simplified extraction technique. Methods: Whole blood was drawn from 32 healthy subjects in standard EDTA blood collection tubes. Total RNA was extracted from whole blood, using the PUREscript RNA Isolation Kit. RT-PCR using intron-spanning primers was used to quantitatively amplify thyroglobulin mRNA, using the ABI PRISM 7700 Sequence Detection System with a fluorescent-labeled, thyroglobulin-specific oligonucleotide probe. Thyroid RNA calibration curves were created using total RNA recovered from a single nondiseased thyroid gland. Results: Qualitative RT-PCR demonstrated the presence of thyroglobulin mRNA in the whole blood sample of each healthy subject. The mean concentration of thyroglobulin mRNA detected in these subjects was 433 ± 69 ng of total thyroid RNA per liter of whole blood (range, 26–1502 ng/L). Overall assay imprecision (CV) was 24% for five samples analyzed 10 times each in separate analytical runs on different days. Conclusions: Thyroglobulin mRNA can be accurately detected and quantified in peripheral blood from healthy subjects. This new quantitative technique may improve the clinical utility of circulating thyroglobulin mRNA detection in patients with thyroid disease.

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