Biomedical subjects
Publications and source records attributed to L M Thienpont.
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We describe the first results of a quantitative LC-tandem mass spectrometry method for urinary C-peptide with the use of [2H14]C-peptide as internal standard. LC was based on gradient elution of a Hypersil PEP C18 column. Mass spectrometry was performed in the negative electrospray ionization mode and by monitoring of the transitions at m/z 1514/1334 ([2H14]C-peptide) and 1507/1320 (C-peptide). For sample preparation, we applied ultrafiltration. The analytical performance of the method in terms of measurement precision gave an RSD of <2% (n=10). The overall imprecision was investigated from independent analysis of two urine samples in six-fold and resulted in an RSD<5%. The limit of detection, expressed as signal-to-noise ratio 3, was approximately 0.15 ng C-peptide injected. Analysis of 10 random urine samples from laboratory volunteers showed interference-free ion chromatograms at a signal-to-noise ratio of approximately 75 on average. The C-peptide concentrations calculated from quantification by the bracketing calibration technique ranged from 32 to 165 ng/ml.
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We investigated the intrinsic (as delivered by the manufacturer) and routine quality of four systems for measurement of serum total magnesium (t-Mg(2+)) by method comparison with an ion chromatography reference method. The results of the study were interpreted on the basis of analytical quality specifications derived from the biological variation of t-Mg(2+), expanded by the analytical uncertainty of the reference measurements. This resulted in limits for systematic error of 2.1% and for total error of 4.3%. The study demonstrated that those limits were challenging for all routine systems. Most of them met the total error criterium just borderline and one showed a considerable systematic error (-5.2%). Concerning the measurement quality in the routine laboratories, the study showed that many were unable to preserve the intrinsic quality of the respective manufacturer. Consequently, loss of system performance in the routine laboratory mostly led to violation of the analytical specifications. Most strikingly, the study revealed enormous quality differences between routine laboratories. This indicates that, still, many routine laboratories do not make adequate use of currently available internal and external quality control tools. Moreover, some laboratories considerably expanded the high end of the reference interval, thereby reducing the diagnostic potential of t-Mg(2+).
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Measurement by ion selective electrode showed that the pH dependency of serum ionised calcium is better described by an inversely S-shaped third-degree function than by the conventionally used logarithmic function.
Isotope dilution-gas chromatography/mass spectrometry (ID-GC/MS) and isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) methods have been developed for an determination of triiodothyronine (T3) in serum and their potential as candidate reference methods investigated. In both methods, (13)C(9)-T3 was used as internal standard. Sample pretreatment consisted of deproteinization, extraction and high performance liquid chromatography (HPLC) purification. Conversion of serum thyroxine (T4) to T3 was controlled by adding (13)C(6)-T4. For GC/MS, T3 and (13)C(9)-T3 were converted to the N,O-di-heptafluorobutyryl (HFB) methyl ester derivatives and monitored at m/z 844 and 853. For LC/MS with electrospray ionization, the transitions m/z 652/661 to 606/614 were monitored. For use of the methods as candidate reference methods, special attention was paid to the calibration and the measurement protocol (duplicate analysis of each sample on three occasions). Evaluation of the ID-GC/MS and ID-LC/MS/MS methods showed the absence of interference by reverse T3 and T4, a limit of detection of 100 pg (GC/MS) and 18 pg (LC/MS), a recovery of 100 +/- 1.5% (95% confidence interval) and good precision (the total coefficient of variation, n = 6, was typically 1.5%). In addition to the recovery study, the accuracy of the methods was proven by method comparison (ID-GC/MS vs. LC/MS/MS) on three sera, showing a maximum deviation of 1.1%. Finally, the ID-GC/MS method was applied for measurement of 10 different human sera with a T3 concentration range from 0.6 to 7.3 ng/mL.
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Reference methods are a key element to the objective of traceability in laboratory medicine. However, to serve this purpose adequately, minimum analytical quality specifications are required. Here, possible strategies for deriving such specifications are presented, being based on concepts developed before by a European Working Group. Distinction is made between "genuine requirements" for reference methods (direct calibration with primary reference materials; absence of sample-related effects) and "performance specifications" (limits for random, systematic and total error, the latter in association with the number of measurements). While the former requirements are considered as conditio sine-qua-non, the latter specifications should be variable, which means that they should be tailored to the specific application of the methods. In general, it is advocated to derive performance specifications for reference methods from desirable specifications of routine methods (analyte-related), although other models should not be ruled out beforehand. Further, it is recommended that reference laboratories make special efforts to demonstrate and maintain a uniform level of quality of reference methods.
We compared the quality of reference measurements for serum potassium in four reference laboratories from three different European countries, using a panel of 60 native patients' samples. The reference methods were based on either ion chromatography (one laboratory) or flame atomic emission spectrometry (three laboratories). Performance specifications for serum potassium measurements were defined as a maximum overall coefficient of variation (CV) of 1.5%, a maximum bias of 0.65% and a maximum total error of 3.0%. The overall imprecision for all laboratories was in the range of 0.7 to 1.3%, and was thus below the proposed specification of 1.5%. However, two laboratories reported 12 and 13 quadruplicates with CVs exceeding this limit. The mean bias (expressed as deviation from the overall mean of all laboratories) for all reference laboratories was < 0.65%. In the lower concentration range, however, one laboratory exceeded this limit. No laboratory measured samples with a total error above 3.0%. From these results, it can be concluded that the reference measurements, and, thus, also the reference methodologies, based on ion chromatography and flame atomic emission spectrometry were equivalent, and able to satisfy current analytical specifications for serum potassium measurements.
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A new candidate reference method is presented for the determination of thyroxine in serum. The method is based on isotope dilution-liquid chromatography/tandem mass spectrometry using electrospray for ionization. The internal standard used was 13C6-thyroxine, sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure, HPLC was performed on a C-18 column with an eluent containing methanol/water/formic acid (60:40:0.1, by volume), and finally thyroxine and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions m/z 777.7--> 731.7 and m/z 783.7--> 737.7, respectively. The detection limit for thyroxine was 6 pg, the within-run coefficient of variation was 1.1%. The samples were measured in six-fold: in duplicate on three independent days. The mean overall coefficient of variation of the method was 1.6%. The new method was evaluated by measuring nine control sera previously determined by an existing ID-GC/MS method. The differences between the results of the two methods ranged from-1.6% to +3.3%, with a mean of +0.2%.
OBJECTIVES: The International Federation of Clinical Chemistry (IFCC) initiated a pilot study, with cortisol as an example, that aims to implement the concept of standardization of hapten immunoprocedures on the basis of metrological traceability. In fact, this standardization concept comes down to correct calibration of measurement procedures, so that measurement results for patient samples can be traced back to the metrologically highest reference of a result, i.e., an SI unit as embodied in the primary reference material. In consequence, demonstration of standardization of a test system on the basis of traceability requires evaluation of measurement results pertaining to patient samples for accuracy. Such an evaluation shall be done by a correlation study of the routine test system with an accuracy-based reference measurement procedure. DESIGN AND METHODS: The IFCC project will select a panel of single donation human serum samples, and assign them with values for cortisol by measurement with at least two isotope dilution-gas chromatography/mass spectrometry reference methods. Limited amounts of the panel will be distributed to manufacturers, with the object to measure the samples with their calibrated immunoprocedures. The patient correlation studies between the routine and the reference methods will then be interpreted in terms of specificity and accuracy. It will also be investigated whether the performed comparison can be used as a basis for re-calibration. From the experience gained through this pilot study for cortisol, IFCC will formulate recommendations and guidance to manufacturers on how to perform and reliably interpret patient correlation studies. In this way, it is to be expected that the project might form a basis for general implementation of the concept of standardization of hapten immunoassays on the basis of metrological traceability.