Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.     

Global trends in quality specifications: a report from the Stockholm Conference

 In Stockholm a conference entitled: "Strategies to Set Global Quality Specifications in Laboratory Medicine" was held in April 1999. The primary aim in organizing the Conference was to provide a vehicle for reaching consensus on global quality specifications in laboratory medicine. This objective was achieved and a lively constructive debate, after the presentations were complete, led to agreement on the principles laid down in the Consensus Statement. The International Federation of Clinical Chemistry (IFCC), the International Union of Pure and Applied Chemistry (IUPAC) and the World Health Organization (WHO) kindly sponsored the Conference but it must be noted that the Consensus Statement reflects the views of the presenters and registrants who participated in the Conference and does not necessarily represent those of the sponsoring bodies. This paper reports on the standardization efforts so far, the Stockholm Consensus Conference, the ISO uncertainty concept and the consensus reality. A hierarchy of quality specifications in laboratory medicine was defined and agreed on. Received: 15 April 2000 · Accepted: 15 April 2000     

Quality management in hematology laboratory improved preanalytics variables

Clinical decisions based on laboratory test values are correctly made when a preanalytical stage is properly performed. In this study we demonstrated that introduction of quality management procedures to Hematology laboratory at Medical Center and Health Care Center have helped to solve problems in the preanalytic stage. Blood clotting is a highly complicated process, which begins during blood withdrawal from vessels. In order to minimize in-vitro changes during collection, transportation and storage, the following steps were followed: centralization of blood collection, strict keeping of time interval between blood withdrawal to testing or freezing and purchasing of adequate freezer for storage. As a result, fibrin clots in frozen plasma were reduced from 10.6% to zero within 13 months and thus, the number of rejected samples decreased. Cooperation with ex-hospital customer who had introduced quality management system and established center for blood collection, separation and distribution have encouraged the introduction of new procedures.     

Role of proficiency testing in monitoring of standardization of hemoglobin A1c methods

After Hemoglobin A_1c (HbA_1c), therapeutic targets for monitoring diabetes therapy were recommended, first, National Glycohemoglobin Standardization Program (NGSP), then, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) developed standardization initiatives. The aim of this article is to demonstrate the role of a proficiency testing (PT) programs in monitoring the long-term effect of these initiatives and the current status of HbA_1c measurement. Measurement precision as a coefficient of variation (CV), measurement bias (bias), and satisfactory HbA_1c result rates in proficiency testing (PT) surveys were evaluated using fresh single donor whole blood PT items and assigned values from a NGSP-certified secondary reference laboratory. Between 2000 and 2010, both CV and bias of the IC measurement method showed a decreasing trend. While the CV of the HPLC measurement method decreased, no significant change was observed in its bias. The rates of satisfactory HbA_1c results in PT surveys were higher in HPLC users than IC users. In 2010, the average CVs in HPLC and IC groups were 2.6 and 3.4?%, biases were 2.7 and 1.8?%, and corresponding total error (TE) estimates were 7.8 and 8.5?%, respectively. These TE values were higher than the maximum permissible measurement error of 7?%, developed based on clinical use of the test. The NGSP and the IFCC networks have promoted improvements in HbA_1c testing; however, tightening of NGSP method certification criteria seems to be necessary to achieve a maximum permissible measurement error of 7?%.     

The influence of blood sample storage time on the propofol concentration in plasma and solid blood elements

In order to describe the changes of propofol concentration in whole blood and in its components during the blood storage we examined venous blood samples collected from patients anaesthetized either with or without propofol. Blood samples from patients anaesthetized without propofol were spike with propofol 45 min before analysis. Propofol concentration was examined in whole blood, plasma, rinsed formed elements and rinsed and lysed formed blood elements by means of HPLC after 1, 4, 7, 13, 21, 25 and 28 days of storage. There was significant decrease in plasma concentration of propofol during the first few days of sample storage followed by its increase during subsequent days. The opposite phenomenon was observed for formed blood elements. The findings support the hypothesis that propofol distribution between blood components changes in time.     

An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer

A flow-injection analytical (FIA) system, comprised of an electrochemical detector with a fructosyl-peptide oxidase (FPOX-CET) reactor and a flow-type spectrophotometer, was proposed for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell. The blood cell samples were hemolyzed with a surfactant and then treated with protease. In the first stage of operation, total hemoglobin in digested sample was determined spectrophotometrically. In the second stage, fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was determined specifically using the electrochemical detector with the FPOX-CET reactor. The FIA system could be automatically processed at an analytical speed of 40 samples per hour. The proposed assay method could determine selectively only the glycated N-terminal residue of β-chain in glycohemoglobin and total hemoglobin in blood cell. The enzymatic hemoglobin A_1c (HbA_1c) value calculated by the concentration ratio of the FVH to total hemoglobin, was closely correlated with the HbA_1c values certified by the Japan Diabetic Society (JDS) and the International Federation of Clinical Chemistry (IFCC).     

Evaluation of diabetic ketoacidosis-related ionic metabolites in human blood serum by capillary electrophoresis.

Three methodologies are presented to contemplate the analysis of sodium, potassium, chloride, bicarbonate, lactate, acetoacetate, and beta-hydroxybutyrate in blood serum samples of diabetic patients using free solution capillary electrophoresis with indirect detection (214 nm for cations and 254 nm for anions). The cations were analyzed in less than 6 min in an electrolyte comprised of 15 mmol x L(-1) imidazole, 5 mmol x L(-1) lactate and 1 mmol x L(-1) 18-crown-6-ether, adjusted to pH 4.5. Chloride and bicarbonate were analyzed in 2 min in a 5 mmol x L(-1) chromate, 0.1 mmol.L(-1) cetyltrimethylammonium bromide (CTAB), pH 9.0 electrolyte solution. Ketoacids and lactate were analyzed in less than 11 min in an electrolyte composed of 15 mmol x L(-1) 3,5-dinitrobenzoate, 0.1 mmol x L(-1) CTAB, at pH 3.5. All methodologies were validated with respect to linearity, selectivity, sensitivity, precision and accuracy performing adequately for clinical purposes.     

当前和下一代锂离子电池电解液的原子尺度微观认识和研究进展

电解液及构筑电极电解液界面对于开发和应用高比容量储能系统至关重要。具体来说，电解液的机械（抗压性、粘度）、热（热导率和热容）、化学（溶解性、活度、反应性）、输运和电化学（界面及界面层）等性质，与其所组成的储能器件的性能直接相关。目前，大量的实验研究通过调控电解液的物理和/或化学组成来改善电解液性能，以满足新型电极材料的工作运行。与此同时，理论模拟方法近年来得到了迅速发展，使人们可以从原子尺度来理解电解液在控制离子输运和构筑功能化界面的作用。站在理论模拟研究的前沿上，人们可以利用其所揭示的机理性认识对新型电解液开展理性设计。本文首先总结了传统电解液的组成、溶剂化结构和输运性质以及电极电解液界面层的形成机理，进一步讨论了利用新型电解液设计稳定电极电解液界面层的方法，包括使用电解液添加剂、高浓电解液和固态电解质，并着重讨论了对这些新型电解液体系进行原子尺度模拟的最新进展，为了解和认识电解液提供更为基本的理解，并为未来电解液的设计提供系统的指导。最后，作者对新型电解液的理论筛选进行了展望。     

Plasma and blood assay of xanthine and hypoxanthine by gas chromatography-mass spectrometry: physiological variations in humans

Plasma and blood xanthine and hypoxanthine levels were assayed using a sensitive and specific method involving gas chromatography-mass spectrometry, associated with an optimized sample preparation procedure. Physiological variation was studied in 224 subjects with no purine metabolism disorders. An age dependency for both compounds was found, comparable with that known for uric acid. The mean plasma levels for the 224 subjects were 0.65 +/- 0.24 microM for xanthine and 1.65 +/- 0.78 microM for hypoxanthine. Corresponding mean blood levels were 0.59 +/- 0.21 microM for xanthine and 1.72 +/- 0.74 microM for hypoxanthine. Plasma and blood levels were significantly different, by ca. 10%. Rapid in vitro release of hypoxanthine from erythrocytes and continuation of intraerythrocytal metabolism lead to overestimation exceeding 10% within half an hour after sample blood collection. Hence samples must be deproteinized promptly. Blood can therefore be conveniently used for oxypurine assay instead of plasma when prompt spinning of samples is difficult to manage, as is usually encountered in clinical practice.     

Pre-cut dried blood spot (PCDBS): an alternative to dried blood spot (DBS) technique to overcome hematocrit impact

Quantification of analytes by Dried Blood Spots (DBS) on different paper cards has been extensively reported in the past several years. However, some factors limit the robustness of the precision and accuracy of DBS such as: hematocrit level, blood viscosity, analyte nature, spotting technique and spotting conditions. As such, the paper material used for DBS must meet strict quality control criteria to produce reliable quantification of drugs: uniformity, no chemical leaching and no chromatographic effect. To overcome these variables, especially the hematocrit impact, a modification of the traditional DBS, named Pre-Cut Dried Blood Spot (PCDBS), is presented. In contrast to the classical DBS technique, the new PCDBS procedure demonstrates no variation in response, within ±3%, independently of the hematocrit level or of the type of card used. The impact of the hematocrit level on the analyte recovery is discussed for both DBS and PCDBS approaches. Moreover, for quantification of naproxen by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), the PCDBS technique was demonstrated to be as precise (%CV ≤3.1%) and accurate (%nominal between 95.4 and 104.4%) as the classical DBS procedure.     

Systematic reviewing in laboratory medicine. Position Paper from the IFCC Committee on Systematic Reviewing in Laboratory Medicine.

Each year a vast number of biomedical articles and books are published and based on the articles reviews are written. Such reviews should be performed in a systematic manner. Systematic reviewing is a new discipline with its own methods for locating, appraising, and summarizing primary studies. Such methods have also been developed for studies on diagnostic test evaluations. It is important for the laboratory disciplines to engage in this work. IFCC has established a Committee for Systematic Reviewing in Laboratory Medicine. This committee will work to promote the understanding, the use and the performance of systematic reviewing.     

Traceability in laboratory medicine

In laboratory medicine meaningful measurements are essential for diagnosis, risk assessment, treatment and monitoring of patients. Thus methods applied in diagnostic measurements must be accurate, precise, specific and comparable among laboratories. Inadequate or incorrect analytical performance has consequences for the patients, the clinicians, and the health care system. One key element of metrology is the traceability of a measurement result to the SI system ensuring comparable results. This principle is described in the ISO/TC 212/WG2 N65 prEN 17511 Standard. In addition to the principles of metrology, the clinical usefulness, the diagnostic needs, and the biological and disease associated variations in patients' specimens have to be considered when the analytical biases for diagnostic purposes are defined. It must be the general goal of diagnostic laboratories to produce results that are true and comparable worldwide. The recent European in vitro diagnostic (IVD) Directive 98/79 EC follows the above mentioned standard of the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN) requesting its application for all IVD reagents used within the European Union. This new European legislation will have a worldwide impact on manufacturers and clinical laboratories and will be implemented in 2003. It states that "traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of a higher order". Thus a worldwide reference system needs to be established by collaboration and mutual recognition between the United States National Institute of Standards and Technology (NIST), European Metrology Institutes (EUROMET), regulatory bodies (e.g. United States Food and Drug Administration, FDA) the IVD industry and professional organizations (e.g. International Federation of Clinical Chemistry and Laboratory Medicine, IFCC). In June 2002, in Paris, representatives of international and regional organizations and institutions decided to form the "Joint Committee on Traceability for Laboratory Medicine" (JCTLM), which will support industry in registration and licensing of the "CE" label to test systems conforming to the IVD Directive.Presented at the International ILAC/IAF Conference on Accreditation in Global Trade, 23–25 September 2002, Berlin, Germany     

Comparison of IFCC and NGSP methods for determination of glycated haemoglobin using advanced regression techniques

The objective of this work was a correct statistical comparison of two assay methods for determination of glycated haemoglobin HbA1c. The immunoturbidimetric determination of HbA1c was performed in two ways: using an automatic analyser Hitachi 912, calibrated according to the IFCC reference system (International Federation of Clinical Chemistry and Laboratory Medicine) and using an analyser Advia 1200 and the NGSP reference system (National Glycohaemoglobin Standardization Program). For statistical comparison of these two analytical methods several advanced regression methods were used, which respect random errors of both compared methods. Specifically, Deming regression with and without weights, orthogonal regression, and Passing-Bablok regression were employed. The results demonstrate that the investigated analytical assay methods do not correspond to each other. The summarized results indicate usefulness of better harmonisation of two existing reference systems.     

Characterization of the cell surface and cell wall chemistry of drinking water bacteria by combining XPS, FTIR spectroscopy, modeling, and potentiometric titrations

Aquabacterium commune, a predominant member of European drinking water biofilms, was chosen as a model bacterium to study the role of functional groups on the cell surface that control the changes in the chemical cell surface properties in aqueous electrolyte solutions at different pH values. Cell surface properties of A. commune were examined by potentiometric titrations, modeling, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. By combining FTIR data at different pH values and potentiometric titration data with thermodynamic model optimization, the presence, concentration, and changes of organic functional groups on the cell surface (e.g., carboxyl, phosphoryl, and amine groups) were inferred. The pH of zero proton charge, pH(zpc) = 3.7, found from titrations of A. commune at different electrolyte concentrations and resulting from equilibrium speciation calculations suggests that the net surface charge is negative at drinking water pH in the absence of other charge determining ions. In situ FTIR was used to describe and monitor chemical interactions between bacteria and liquid solutions at different pH in real time. XPS analysis was performed to quantify the elemental surface composition, to assess the local chemical environment of carbon and oxygen at the cell wall, and to calculate the overall concentrations of polysaccharides, peptides, and hydrocarbon compounds of the cell surface. Thermodynamic parameters for proton adsorption are compared with parameters for other gram-negative bacteria. This work shows how the combination of potentiometric titrations, modeling, XPS, and FTIR spectroscopy allows a more comprehensive characterization of bacterial cell surfaces and cell wall reactivity as the initial step to understand the fundamental mechanisms involved in bacterial adhesion to solid surfaces and transport in aqueous systems.     

Determination of the aminoglycoside antibiotics sisomicin and netilmicin in dried blood spots on filter discs, by high-performance liquid chromatography with pre-column derivatization and fluorimetric detection

A simple method for the determination of the aminoglycoside antibiotic sisomicin or its 1-N-ethyl derivative, netilmicin in whole blood, using dried blood spots (DBSs) on filter-paper punched discs has been developed. Sisomicin or netilmicin in the DBSs were recovered most effectively in 0.5 M Na2HPO4 using ultrasonication. The eluates from the DBSs were treated by ultrafiltration for deproteinization and subjected to pre-column fluorescent derivatization using o-phthalaldehyde and beta-mercaptopropionic acid in 0.05 M KH2PO4-borate buffer (pH 9.0), followed by determination by reversed-phase high-performance liquid chromatography. The detection limits of sisomicin and netilmicin in the DBSs on punched discs (10.1 microliters of whole blood) were 0.053 and 0.50 micrograms per ml of whole blood, respectively (signal-to-noise ratio greater than or equal to 2). The method permits a simple collection of blood at the microlitre level and should prove particularly useful for monitoring sisomicin and netilmicin in blood at therapeutic levels in geriatric and paediatric patients.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes.

Red blood cells contain a protein that activates membrane-bound (Ca2+ + Mg2+)-ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175--4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Gas chromatographic headspace analysis of sevoflurane in blood.

We have developed a rapid, simple and precise gas chromatographic headspace analysis for sevoflurane in blood which circumvents problems associated with the high volatility and low blood/gas partition coefficient of this anesthetic drug. Blood standards are easily prepared by volumetric addition of a saturated aqueous solution of sevoflurane. Likewise, internal standardization is achieved using a saturated aqueous solution of halothane. Chromatographic conditions are similar to those commonly used for the analysis of blood ethanol. A simple method is also described for the preparation of stable and precise, aliquots of quality control materials for this assay.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

Operating mechanism of the electrolyte cathode atmospheric glow discharge

Cathode fall ( U(cf)), cathodic current density and atomic emission intensities originating from metal salts in the electrolyte cathode were measured as a function of different discharge parameters. Emission intensities in function of cathode fall indicate a potential barrier in the sputtered mass flux. This means that the primary particles of the cathode sputtering are of positive charge and the cathode fall including its internal variables is the most important factor. The measured current density and the U(cf) as a function of pressure are in accordance with the low pressure data in the literature. The observed decrease of the U(cf) with decreasing pH was explained by a model in that the secondary electron emission coefficient of the cathode (gamma) is controlled through a reaction net of competing reactions of different electron scavengers involving the hydroxonium ions of the cathode solution. The model revealed two different electron emission processes of the electrolyte cathode, an emission coupled with hydrated electrons is dominating below pH 2.5 while a proton-independent emission of poor efficiency is working above pH 3. Our model fits to the reported yields of the ultimate products both in the solution and in the gas phase and offers a calculation of gamma and U(cf) in the function of the cathode acidity. The model provides two other independent gamma calculation methods based on product analysis data.     

Copper adsorption and Si, Al, Ca, Mg, and Na release from clinoptilolite

Copper adsorption onto clinoptilolite (natural zeolite), Al/Si dissolution, and Mg, Ca, and Na release from the substrate were the subjects of the investigation described here. Experimental variables were Cu and electrolyte concentrations and solution pH. Copper adsorption was found to increase with increased pH and with decreased electrolyte concentration. Large amounts of K were also adsorbed from electrolyte. Since solution pH was assumed as a variable, the effects of [H(+)] differentiation on Cu adsorption and on Al/Si dissolution were also examined. Al dissolution was affected mainly by electrolyte concentration, whereas Si dissolution was affected mainly by adsorbed Cu amount. It was assumed that the release of Mg, Ca, and Na occurs through ion-exchange reactions with solution K(+), because their release is affected more by electrolyte concentration than by adsorbed Cu. From the study of FTIR spectra for various samples used in the present investigation, we observed that the removal of framework Si/Al shifts the band which was attributed to O-T-O stretching vibration toward higher frequency. Significant changes were observed for the bands assigned to Si-OH-Al bridges and to monomeric and polymeric hydrogen bonds at the region between 3650 and 3200 cm(-1). It is proposed that the Cu species caused the destruction of H-bonded structures, whereas K adsorbed species were located at exchangeable sites after an ion-exchange process between K and Ca, Mg, and Na from the zeolite's surface. An expansion of the zeolite framework was detected from XRD patterns under acid conditions.