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.

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.

Determination of glutathione and glutathione disulfide in human whole blood using HPLC with coulometric detection: A comparison with fluorescence detection

2011 ◽  
Vol 76 (4) ◽  
pp. 277-294 ◽  
Author(s):  
Roman Kanďár ◽  
Pavla Žáková ◽  
Miroslava Marková ◽  
Halka Lotková ◽  
Otto Kučera ◽  
...  
Keyword(s):  
Detection Limit ◽  
Whole Blood ◽  
Trichloroacetic Acid ◽  
Signal To Noise Ratio ◽  
Glutathione Disulfide ◽  
Simple Method ◽  
Human Whole Blood ◽  
Coulometric Detection ◽  
Coefficients Of Variation

We describe a relatively simple method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human whole blood. We have used an HPLC with coulometric electrochemical detection for the simultaneous measurement of GSH and GSSG. Diluted and filtered trichloroacetic acid extracts were injected directly into the HPLC system and were eluted isocratically on a Polaris 5u C18-A, 250 × 4.6 mm analytical column. Glutathione in samples extracted with trichloroacetic acid and diluted with 1.0 mMhydrochloric acid was stable at 4 °C for at least 8 h. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked whole blood samples were at intervals 91.6–97.6% for GSH and 85.0–104.4% for GSSG. The linear range is 5.0–2000.0 μmol/l, with a detection limit of 2.1 μmol/l (signal-to-noise ratio = 3) for GSH and 2.0–250.0 μmol/l, with a detection limit of 0.9 μmol/l for GSSG.

scholarly journals High Dose (5mg) Daily Folic Acid Supplement in Healthy Brazilian Volunteers Increases Mononuclear TNF-α Expression and Reduces NK Cell Number and Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4531-4531
Author(s):  
Juliano Bertinato ◽  
Clovis Paniz ◽  
Maylla Rodrigues Lucena ◽  
Patrícia Mendonça da Silva Amorim ◽  
Guilherme Wataru Gomes ◽  
...  
Keyword(s):  
Folic Acid ◽  
Nk Cells ◽  
Mrna Expression ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Nk Cell ◽  
Interferon Γ ◽  
Cell Number ◽  
Interleukin 8 ◽  
Tnf Α

Abstract Background In Brazil, wheat and corn flour is fortified with 150 µg of folic acid (FA), the synthetic form of folate. Individuals with increased cell duplication, including pregnant women and patients with hemolytic anemia need increased amounts of folate. The effects of amounts of FA higher than the defined tolerable upper intake of 1 mg/day are poorly understood. Some Brazilian patients with hemolytic anemia, such as hereditary spherocytosis (HS), have been receiving 5mg/day supplemental FA, in addition to being exposed to mandated food fortification with FA. Our previous data has shown that patients with HS have higher serum folate levels than healthy controls, as well as higher mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes (1). However, it was not clear whether the increased mRNA expression resulted from folic acid use or underlying disease. Objective The aim of this study was to verify the effects of an intervention with 5mg/day FA on folate levels (serum and whole blood), serum inflammatory markers levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes and cytotoxicity of NK cells in healthy Brazilian volunteers. Material and methods Fifteen male and fifteen female healthy subjects were given 5mg/day FA for 90 days. Blood was collected at baseline, day 45 and day 90 for blood count, including reticulocytes, C-reactive protein and lactate dehydrogenase (LDH). Folate (serum and whole blood) and vitamin B12 were determined by a microbiological method. Serum cytokines levels were measured using a Milliplex Map kit. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed using Real Time PCR. Cytotoxicity of lymphocytes and NK cell number were measured by flow cytometry. Results All blood count parameters were unaffected by FA intervention, whereas there was a slight increase in concentrations of LDH (P = 0.001) after 90 days compared with baseline and 45 day measurements. The folate levels (serum and whole blood) were higher at 45 and 90 days of intervention with 5mg/day of FA (P<0.001 for both). There were no differences among the basal and the follow up for serum vitamin B12, total homocysteine, cytokines IL6, IL8, IL10, IFNγ and TNFα levels (P>0.05). The mRNA expression of IL8 was higher at 45 days of intervention (Fig 1), while mRNA expressions of TNF-α were elevated at 45 and 90 days compared with baseline (Fig 1). No difference was found in mRNA of DHFR, MTHFR and IFNγ in this study. The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups After 5 mg FA daily there was a reduction in the number and cytotoxic capacity of NK cells (Table 1). The data are median and interquartile intervals. The groups were compared using the Friedman test, when significant was performed for multiple comparisons Dunn`s test. Different letters show significant differences between groups. Conclusions Intervention with 5 mg/day of FA in healthy people was associated with around 4-fold increase in serum and whole blood folate, accompanied by increased mRNA expression of proinflammatory cytokines IL8 and TNF-α and a reduction in NK cell number and cytotoxicity. High dose FA fortification may result in changes in innate immune parameters that could perturb immune surveillance pathways. Financing: FAPESP 2012/12912-1, CNPq 4826412012-6 and CNPq 401586/2014-6 References 1. Paniz, C et al. Blood 2014;124:4005. Presented at the ASH 2014. Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Figure 1. mRNA expression of IL8 (A) and TNFα (B) genes in healthy subjects before and 45 and 90 days after 5 mg FA daily Tabel 1 Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Tabel 1. Number, lytic activity and cytotoxic capacity of NK cells after intervention with 5 mg/day of folic acid Disclosures No relevant conflicts of interest to declare.

Novel LC–MS/MS method for the determination of selumetinib (AZD6244) in whole blood collected with volumetric absorptive microsampling

Bioanalysis ◽  
2020 ◽  
Vol 12 (13) ◽  
pp. 883-892
Author(s):  
Ramakrishna R Voggu ◽  
Theodore S Brus ◽  
Chineta T Barksdale ◽  
Paul Severin ◽  
Patricia Hansen ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ammonium Hydroxide ◽  
Bioanalytical Method Validation ◽  
Simple Method ◽  
Human Whole Blood ◽  
Regulatory Guidance ◽  
Validation Parameters ◽  
Diagnostic Applications ◽  
Volumetric Absorptive Microsampling

Aim: A method has been developed and validated for quantitation of selumetinib in human whole blood collected using a Mitra™ volumetric absorptive microsampling device. This device is patient-friendly, affording less-invasive sampling with broad applicability to clinical and diagnostic applications – specifically in pediatric populations. Materials & methods: In this method, drug is extracted from the Mitra device via sonication in methanol: Ammonium hydroxide, then analyzed by LC–MS/MS. The linear range for selumetinib analysis is 2.00–2000 ng/ml. Results: All validation parameters met acceptance criteria established in agreement with current regulatory guidance for bioanalytical method validation. The stability of selumetinib in Mitra tips was established at both ambient and frozen conditions. Conclusion: A simple method has been developed and validated for determination of selumetinib from human whole blood, collected using volumetric absorptive microsampling and analyzed by LC–MS/MS.

Problems in extraction and spectrophotometric determination of chlorophyll from epilithic microbial biofilms: towards a standard method

1999 ◽  
Vol 79 (3) ◽  
pp. 551-558 ◽  
Author(s):  
R.C. Thompson ◽  
M.L. Tobin ◽  
S.J. Hawkins ◽  
T.A. Norton
Keyword(s):  
Standard Method ◽  
Relative Efficiency ◽  
Room Temperature ◽  
Standard Procedure ◽  
Suspended Material ◽  
Sample Collection ◽  
Simple Method ◽  
Microbial Biofilms ◽  
Epilithic Biofilms

A variety of methods are available to extract chlorophyll from epilithic biofilms using solvents. The relative efficiency of these has not been determined simultaneously and there is no recognized standard procedure. In this paper techniques for sample collection, storage, preparation and extraction are reviewed and compared experimentally.Extraction of chlorophyll was incomplete unless biofilms were fully hydrated. This factor was highly significant for all the solvents tested, with at least three times more pigment being extracted from hydrated samples than from dry ones. Methanol was the most efficient solvent, releasing over 96% of the total chlorophyll during a single extraction; hot ethanol extracted 86%, while acetone extracted less than 50%. Sonicating samples during extraction did not release any additional pigment. Centrifuging to remove suspended material did not alter estimates and was not advantageous. Rugose rock surfaces released more chlorophyll than smooth ones. However, a simple method to quantify surface rugosity at an appropriate scale was not available.Based on these observations, a standard method for chlorophyll extractions from epilithic biofilms using 100% methanol at room temperature (20°C) is proposed. This technique requires considerably less supervision than previously preferred methods and gave a chlorophyll extract which was stable for 15 h.

Determination of plasma and erythrocyte lithium concentrations by atomic absorption spectrophotometry.

1977 ◽  
Vol 23 (1) ◽  
pp. 41-45 ◽  
Author(s):  
G H Hisayasu ◽  
J L Cohen ◽  
R W Nelson
Keyword(s):  
Atomic Absorption ◽  
Whole Blood ◽  
Room Temperature ◽  
Clinical Laboratory ◽  
Lithium Concentration ◽  
Atomic Absorption Spectrophotometry ◽  
Laboratory Procedure ◽  
Absorption Spectrophotometry ◽  
Routine Clinical Laboratory

Abstract We describe a method for determining plasma and erythrocyte lithium concentrations by atomic absorption spectrophotometry. Plasma and hemolyzed whole-blood are diluted and analyzed, with use of a lithium hollow-cathode lamp, at 670.8 nm. Erythrocyte lithium concentrations are calculated indirectly from the hematocrit. The standard deviation for a 0.43 mmol/liter pool of whole blood, run daily over 11 months, was +/-20 mumol/liter (CV=5.1). The lithium concentration of a lyophilized pool assayed periodically over the same period (n=127) was 1.84+/-0.05 mmol/liter (CV=2.7%). The relatively low erythrocyte/plasma lithium ratio and the microhematocrit centrifugation force (9600 to 13 600 X g) make corrections for trapped plasma insignificant. Problems with matrix matching and viscosity are overcome by using a plasma pool standard for calculations. Values for erythrocyte lithium concentrations were unchanged in samples stored at room temperature up to 24 h. Hemolysis appears to be of possible clinical significance. This method is useful as a routine clinical laboratory procedure for monitoring patients with affective disorders, who are undergoing therapy with lithium.

Determination of sodium and potassium with ion-selective electrodes.

1984 ◽  
Vol 30 (3) ◽  
pp. 433-436 ◽  
Author(s):  
N Fogh-Andersen ◽  
P D Wimberley ◽  
J Thode ◽  
O Siggaard-Andersen
Keyword(s):  
Whole Blood ◽  
Venous Blood ◽  
Ion Selective Electrodes ◽  
Liquid Junction ◽  
Sample Handling ◽  
Blood Ph ◽  
Ionic Binding ◽  
Sodium And Potassium ◽  
Junction Potential

Abstract We compared different sample-handling techniques for measurement of Na+ and K+ with ion-selective electrodes (ISE). Imprecision was less for venous blood (with a minimum of heparin) than for plasma, serum, or capillary blood. The results for K+ were higher for serum than for whole blood, and higher for whole blood than for plasma. The latter difference was apparently due to release of K+ during the analysis. Values were more stable for whole blood stored at 20 degrees C than at 4 degrees C or 37 degrees C. The molality of Na+ in the plasma of mixed whole blood changed by -10.5 mmol/kg per unit change in blood pH. This could be explained by the different H+ buffering capacities of plasma and erythrocyte fluid, because when the pH is changed, the concentration of small anions in erythrocytes changes more than it does in plasma, with a consequent osmotic movement of water across the erythrocyte membrane. When we took into account the residual liquid-junction potential and the mass concentration of water in each of 65 patients' sera, the molality determined for Na+ was 1% lower and that of K+ 3% lower by ISE than by flame photometry--differences that may be related to ionic binding or to a lower molal activity coefficient in serum than in the calibrator.

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