Glycated hemoglobin (HbA1c) measurement in frozen whole blood depends on baseline values of fresh samples

Glycated hemoglobin (HbA1c) has been recently adopted as a diagnostic marker of type 2 diabetes. However, its usage is currently limited to fresh blood samples. To allow retrospective HbA1c measurement in blood banks developed in large epidemic studies, here, we contribute to validate HbA1c assessment in frozen versus fresh blood samples from a cohort of diabetic/nondiabetic adult subjects. HbA1c was measured by HPLC in 237 fresh whole blood samples and on the same samples after a 12-month storage and a further 6-month-refrozen storage. Mean HbA1c?±?SD in fresh, frozen, and refrozen samples was 6.9?±?1.2, 6.6?±?1.1, and 6.4?±?1.0 % for the Diabetes Control and Complications Trial and 52?±?13, 49?±?12, and 46?±?11 mmol/mol for the International Federation of Clinical Chemistry and Laboratory Medicine reference, respectively. A significant correlation was found between fresh/frozen and fresh/refrozen (R?=?0.994 and 0.993, P?<?0.001) samples. HbA1c relative error ratio (%RER) between frozen/refrozen and fresh samples significantly correlated with HbA1c and depended on fresh value range, increasing in the five HbA1c classes (<6.0, 6.0–6.5, 6.5–7, 7–8, ≥8 %, corresponding to <42, 42–48, 48–53, 53–64, ≥64 mmol/mol, P?<?0.001). In particular, the 6.5 % (48 mmol/mol) HbA1c diagnostic cutoff of fresh samples identified two classes reflecting significant differences in %RER (2.8?±?2.0 and 3.3?±?1.7; P?<?0.05) between frozen and fresh samples. In conclusion, our results demonstrate a high correlation between data from fresh and frozen samples, with a very limited %RER between the two measurements, which increases with baseline HbA1c levels. Accordingly, when analyzing biobank frozen specimens for diagnostic purpose, the effect of the HbA1c range should be taken into account.

Development of an electrophoretic method based on nanostructured materials for HbA1c determination

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 μg/mg) as pseudo‐stationary phase. Before the sample injection, SiO₂NP─B(OH)₂ were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%⁻¹ × HbA1c − 377.02; R² = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t‐test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.     

Development of an electrochemical immunosensor for the detection of HbA1c in serum

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.     

Considerations with regard to the calculation of key comparison reference values and uncertainties

Much has been published over the last years on finding the best (consensus) reference value for a given set of key comparison data, and a suite of approaches proposed. In this paper, several aspects are highlighted which should be taken into account when deciding on key comparison strategies, protocols, and the best approach to reliable key comparison reference value and uncertainty. Some of these aspects are briefly discussed. The paper is intended to intensify discussions, and any comments and opinions are welcome. Presented at the CCQM 2008 KCRV Workshop, April 2008, Sèvres, France. Papers published in this section do not necessarily reflect the opinion of the Editors, the Editorial Board and the Publisher.     

The traceability chain in medical laboratories: a long and winding road, the example of HbA1c

In medical laboratories, a metrological basis for quantitation is missing for many tests. This is recognised by the profession and over the last decade the concept of metrological traceability is advocated. However, daily practice is refractable: for many analytes reference measurement procedures are missing and once in place, clinicians obstruct the new units. This wearisome process is described in general terms and illustrated by the specific case of HbA_1c. A working group of the scientific division of the IFCC developed a reference system for this key parameter in diabetes management. This system is worldwide accepted as the analytical anchor. However, clinicians objected the new units. A debate of years followed. This urged the international diabetes organisations IDF, EASD and ADA to find a solution to avoid confusion. They came up with a consensus statement, but this compromise—report of HbA_1c in three units—was unpractical and not accepted. Finally, decisions were made at a national level: the European Union and Japan will report in the new units, the US in their old national units and an additional unit, the estimated average glucose.     

基于对氨基苯硼酸修饰磁性纳米粒子及丝网印刷技术测定糖化血红蛋白

制备了对氨基苯硼酸修饰的磁性纳米粒子,其与血液中的糖化血红蛋白(Hb A1c)结合,通过网状玻璃态碳(RVC)电极的磁性区域后,可实现与未糖基化的血红蛋白分离。通过壳聚糖(CS)、正硅酸乙酯(TEOS)和碳纳米管构成的溶胶-凝胶膜修饰丝网印刷电极对血红蛋白进行电化学检测,从而建立了一种用于检测糖化血红蛋白的新方法。在0.10 V电位下,丝网印刷电极的电量与Hb A1c和Hb的浓度有较好的线性关系,检出限分别为6 mg/m L和0.05 mg/m L。该方法电极制作简单,有较好的重现性和稳定性,且能有效排除抗坏血酸(AA)、尿酸(UA)等的干扰,已成功应用于实际样品中Hb A1c的检测。     

Development of an ICP-IDMS method for accurate routine analyses of toxic heavy metals in polyolefins and comparison with results by TI-IDMS

An inductively coupled plasma isotope dilution mass spectrometric (ICP-IDMS) method was developed as a suitable method - with respect to its sensitivity, precision, accuracy, and time-consumption - for the analysis of toxic heavy metal traces (Pb, Cd, Cr, and Hg) in polyolefins. Results for Pb, Cd, and Cr were compared with those obtained by thermal ionization isotope dilution mass spectrometry (TI-IDMS), which was used as a reference method. Because of its high first ionization potential and its high volatility mercury could not be determined by TI-IDMS. A multi-element spike solution, containing isotopically enriched 206Pb, 116Cd, 53Cr, and 201Hg, was used for the isotope dilution step. Decomposition of the polyolefin samples was carried out with concentrated HNO3 at temperatures of about 300 degrees C in a high pressure asher (HPA). This procedure decomposes polyolefins completely and allows isotopic equilibration between sample and spike isotopes. Detection limits of 16 ng/g, 5 ng/g, 164 ng/g, and 9 ng/g were obtained for Pb, Cd, Cr, and Hg by ICP-IDMS using only sample weights of 0.25 g. In different commercially available polyethylene samples heavy metal concentrations in the range of < 5 ng/g to 4 x 10(3) ng/g were analyzed. Both mass spectrometric methods were applied within the EU project "Polymeric Elemental Reference Material (PERM)" for the certification of two polyethylene reference materials. The ICP-IDMS results agreed very well with those of TI-IDMS which demonstrates the accuracy of the ICP-IDMS method also suitable for routine analyses.     

Development of an ICP-IDMS method for accurate routine analyses of toxic heavy metals in polyolefins and comparison with results by TI-IDMS

An inductively coupled plasma isotope dilution mass spectrometric (ICP-IDMS) method was developed as a suitable method – with respect to its sensitivity, precision, accuracy, and time-consumption – for the analysis of toxic heavy metal traces (Pb, Cd, Cr, and Hg) in polyolefins. Results for Pb, Cd, and Cr were compared with those obtained by thermal ionization isotope dilution mass spectrometry (TI-IDMS), which was used as a reference method. Because of its high first ionization potential and its high volatility mercury could not be determined by TI-IDMS. A multi-element spike solution, containing isotopically enriched ²⁰⁶Pb, ¹¹⁶Cd, ⁵³Cr, and ²⁰¹Hg, was used for the isotope dilution step. Decomposition of the polyolefin samples was carried out with concentrated HNO₃ at temperatures of about 300?°C in a high pressure asher (HPA). This procedure decomposes polyolefins completely and allows isotopic equilibration between sample and spike isotopes. Detection limits of 16 ng/g, 5 ng/g, 164 ng/g, and 9 ng/g were obtained for Pb, Cd, Cr, and Hg by ICP-IDMS using only sample weights of 0.25 g. In different commercially available polyethylene samples heavy metal concentrations in the range of < 5 ng/g to 4 × 10³ ng/g were analyzed. Both mass spectrometric methods were applied within the EU project “Polymeric Elemental Reference Material (PERM)” for the certification of two polyethylene reference materials. The ICP-IDMS results agreed very well with those of TI-IDMS which demonstrates the accuracy of the ICP-IDMS method also suitable for routine analyses.     

Considering uncertainty of measurement when assessing compliance or non-compliance with reference values given in compositional specifications and statutory limits: a proposal

Considering the uncertainty of measurement (UOM) is mandatory when assessing compliance with reference values given in compositional specifications and statutory limits, but the matter is still open to question. Working in the signal or concentration domains and considering false negative together with false positive errors are the main points of debate. Frequently, the available approaches look too simplified for being accurate or too complex (since more rigorously formulated from a theoretical point of view) for being largely acceptable. In the Authors opinion, assessing compliance with reference values given in compositional specifications and statutory limits is a problem similar to that of estimating the limit of detection. This allows proposing a simple operational approach based on well-known and accepted assumptions and approximations. This proposal, described in the light of the most recent literature information, is aimed to stimulate a critical discussion in view of evaluating possible corrections to the generally accepted approach.Papers published in this section do not necessarily reflect the opinion of the Editors, the Editorial Board and the Publisher.     

Recent advances in ratio primary reference measurement procedures (definitive methods) and their use in certification of reference materials and controlling assigned values in proficiency testing

Three very accurate (definitive) methods by RNAA for the determination of Se, As and Fe respectively, which were recently elaborated in our laboratory, are reviewed and their use in certification of reference materials and in checking the assigned values in proficiency tests is demonstrated on several examples. According to VIM 3 nomenclature these methods may be called: ratio primary reference measurement procedures (RPRMPs). RPRMPs with their expanded uncertainties of 2.7–3.6 % are comparable to ID-MS methods and are the only methods of such high metrological quality which can be used for the determination of trace amounts of monoisotopic elements.     

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).     

Measurement of PAHs in environmental matrices: results from an interlaboratory comparison on the different steps of the measurement procedure

Proficiency testing (PT) is becoming a feature of laboratory accreditation and the PT results are used to assess the technical competence of the participating laboratories. ISPRA (former APAT) plays an important role in supporting the Italian laboratories belonging to the network of the Regional Environmental Agencies to improve the quality of their analytical measurements. As a consequence, ISPRA organized an interlaboratory comparison to assess the performance of the laboratories on PAH measurement procedure. The interlaboratory comparison was drawn separating the different steps of the measurement (from the extraction to the instrumental measurement). Two matrix reference materials: (1) a polluted soil and (2) an extract reference material of the same polluted soil and a “blind” PAH mixture standard stock solution were distributed to 59 Italian laboratories. The results of interlaboratory comparison showed a significant dispersion of the PAH measurements that masks the effects of the different extraction and cleanup procedures used, but it is consistent with the results of other European interlaboratory comparisons.     

Rapid method for determination of protein content in cereals and oilseeds: validation, measurement uncertainty and comparison with the Kjeldahl method

The objective of this research was to test suitability of the Dumas combustion method to completely substitute the Kjeldahl method in routine laboratory determination of crude protein content in cereals and oilseeds. The validation of the method demonstrated that it is able to determine crude protein content in cereals and oilseeds in an efficient and accurate manner, with a detection limit w(N) = 0.006%, quantification limit w(N) = 0.019%, repeatability precision RSD _r = 0.41%, intra-laboratory reproducibility precision RSD _R = 0.74%, trueness, expressed in terms of bias b = 0.43%, and linear response between (2.36–19.2) mg N. Measurement uncertainty, expressed as relative expanded uncertainty (coverage factor k = 2, confidence level 95%), was calculated from validation data (U _rel = 2.24%). In order to examine the relationship between two methods, 15 cereal grain and oilseed samples were analyzed using Dumas and Kjeldahl procedure. The Kjeldahl procedure gave slightly lower w(N) values than the Dumas procedure: w _K(N) = 0.9905 w _D(N) = 0.0376 (R ²  = 0.9996). Relative standard deviations and results of homogeneity test obtained during analysis of complex cereal products (cereal breakfast and muesli bars) show that the Dumas combustion method may be less suitable for analysis of such samples compared to Kjeldahl method.     

Comparability and recognition of chemical measurement results – an international goal

As a consequence of the globalisation of trade and industry and other human activities, reliability of and confidence in measurement results is increasingly required, also in the field of chemical analysis, so that measurements made in one country will be accepted in other countries without the necessity to repeat them. The prerequisite for confidence is comparability on the basis of known uncertainties which in turn are based on traceability to recognised references. Traceability structures for chemical measurements are required which, by providing calibration means traceable to national standards, allow uncertainty statements to be made at field level, thus establishing comparability. Such traceability structures are now being developed in all industrialised countries. To ensure international comparability, mutual recognition of the national activities in metrology in chemistry is required in addition. The Mutual Recognition Agreement (MRA) for national measurement standards and calibration certificates issued by national metrology institutes, which is currently under way within the framework of the Metre Convention, aimes at providing the necessary international confidence for all kinds of measurements. The field of chemical analysis is included in the international metrological infrastructure through the new Consultative Committee for Amount of Substance (CCQM). Carefully selected key comparison measurements, which cover the most important areas where traceability is required, and which are carried out by national metrology institutes in cooperation with other national institutes entrusted with the provision of part of the national references for chemical measurements, form the basis for declarations of equivalence under the MRA. The results of the first key comparisons and studies carried out so far clearly show that the group of laboratories involved in the key comparisons is capable of establishing the international references (key comparison reference values) for chemical measurements with sufficient accuracy, also in complicated matrices.     

Systematic kinetics study of FoF1-ATPase: analytic results and comparison with experiments

The systematic kinetics of the holoenzyme F oF 1-ATPase has been investigated by a tri-site filled, random binding order, and stochastic mechanochemical tight coupling model. The connection between the mechanical rotation speed and the chemical quantities such as the concentrations of substrates, the proton motive force, and the mechanical damping coefficient, has been analytically derived. The enzymatics based on ensemble experiments and single-molecule assays can be discussed systematically. Our model predictions agree well with both ensemble and single-molecule experimental results. Furthermore, this model can be used to study the dynamics of F oF 1-ATPase in a vesicle system.     

Field measurement comparison of aerosol metals using aerosol beam focused laser-induced plasma spectrometer and reference methods

Several tests were conducted in July 2002 on an instrument developed by researchers at the Oak Ridge National Laboratory (ORNL) for in-situ, real-time measurement of metal-laden aerosol particles in flue gas from combustion facilities. Stock aqueous solutions of prepared metal concentrations were nebulized and injected into the flue gas to provide spikes for the instrument testing. Strengths of the solutions were designed such that a reference method (RM) was able to obtain a sufficient amount of material on filter samples in 30 min cycles; the collected filters were subsequently analyzed in an analytical laboratory using certified methods. Parallel aerosol measurements were performed by one operator using the ORNL instrument. The recorded signal of an individual element was analyzed using the Lorentzian model and the concentration calculated from a calibration curve that was established prior to the experiments. RM data were able to reflect the loads simulated in the spiked waste stream. However, data collected using the RM was unable to reflect the transient load condition in the flue gas and missed a sample containing beryllium. The possibility of bias in the RM determination of chromium could exist. With the real-time detection capability, the instrument was able to reveal the transient emission variation by making seven consecutive measurements within a 30 min cycle. The measurements for mercury by both techniques appeared to be in good agreement. Further improvement of the system includes the development of user-friendly software for modeling, data analysis tools, and packaging suitable for field transportation.