Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.

The TGFß family member myostatin (growth/differentiation factor-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin, and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with the Compact genetic background and nonmutant myostatin with the wild-type background, we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism, and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compacts, the absolute size of heart and kidney is not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity and improves whole body glucose uptake, insulin sensitivity, and 18FDG uptake of skeletal muscle and white adipose tissue, whereas the Compact genetic background has the opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed, leading to altered activation of Smad2, Smad1/5/8, and Akt, and an increased level of p-AS160, a Rab-GTPase-activating protein responsible for GLUT4 translocation. Based on our analysis, the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but those can compensate for each other when systemic metabolic effects are compared.

Diabetes mellitus: the long way of standardization of HbA(1c) to the level of highest metrological order.

Glycated haemoglobin (HbA(1c)) measurements are used in clinical studies and for the management of diabetic patients. Various efforts were made to standardize the HbA(1c) measurements with consensus standards and standards based on a reference measurement procedure with external calibration. According to ISO 17511 a standard should meet highest accuracy possible, have a defined uncertainty of measurement and the calibration should be traceable to SI units. For HbA(1c) this has been realized using a LC-ID-MS procedure based on the existing reference measurement procedure.

Procedure for determination of immunosuppressive drugs in whole blood with liquid chromatography-isotope dilution mass spectrometry.

BACKGROUND: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS). From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required. METHODS: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1% v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosuppressive drugs was performed by isotope-dilution mass spectrometry using [2H12]-Cyclosporin A [13C, 2H3]-Rapamycin, [13C, 2H2]-Tacrolimus, and [13C2, 2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards. RESULTS: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5% for cyclosporin A, 101.6 - 103.0% for sirolimus, 100.0 - 101.2% for tacrolimus, and 99.5 - 102.4% for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60% for cyclosporin A in the concentration range between 8.1 and 979 microg/L, 0.92 - 1.72% for sirolimus in the concentration range between 2.1 and 33.2 microg/L, 0.44 - 1.06% for tacrolimus in the concentration range between 2.0 and 30.8 microg/L and 0.82 - 4.34% for everolimus in the concentration range between 2.1 and 31.4 microg/L. CONCLUSIONS: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.

Liquid chromatography-isotope dilution-mass spectrometry as a new basis for the reference measurement procedure for hemoglobin A1c determination.

BACKGROUND: Standardization of hemoglobin (Hb)A(1c) measurements is a process of considerable interest for quality assurance in diabetes management. To contribute to continuous quality improvement and fulfillment of the requirements for reference measurement procedures according to the standards of the International Organization for Standardization, we developed a calibration system of highest metrological order using isotope dilution-mass spectrometry with a reference material. METHOD: Samples were prepared by enzymatic cleavage based on the IFCC reference measurement procedure for LC-MS analysis. After digestion the samples were spiked with [D7]-labeled glycated and nonglycated hexapeptides as internal standards for quantification. LC-MS analysis was performed by using a C12 reversed-phase column and a gradient of acetonitrile/H(2)O containing 0.1% formic acid. RESULTS: Calibration systems for HbA(1c) determination based on liquid chromatography-isotope dilution-mass spectrometry (LC-ID-MS) and on the IFCC reference measurement procedure were compared. A linear regression analysis demonstrated a correlation of r(2) = 1.00 between the 2 different calibration systems. Mean deviation was 5.5% for the calibration and 3.3% for hemolysate samples, with a mean expanded uncertainty of 4.9%. CONCLUSIONS: This LC-ID-MS procedure allows the current IFCC reference measurement procedure for HbA(1c) to be raised to a higher order of accuracy.

The alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood--a candidate reference measurement procedure.

The article describes the development and evaluation of the alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood without the need for toxic materials and suitable for use in laboratories in countries with limited resources and restricted import of toxic materials. The validation of the method has been performed in accordance with the requirements set out in the international standard ISO 15193, which describes the procedures necessary for development of a candidate reference measure-ment procedure. The main results were: The trueness of the haemiglobin cyanide (HiCN) method depends upon the diluent used. The international haemiglobin cyanide reference material BCR CRM 522 does not have the same chemical properties as that derived from fresh lysed erythrocytes and cannot be used for calibrating the AHD575 method. The transformation of oxyhaemoglobin to haematin at pH 13 proceeds 5-8 times more rapidly than its conversion to haemiglobin cyanide. The AHD575 method yields results comparable with the HiCN method and uses a readily available crystalline standard of high purity. The introduction of the AHD575 method does not require new reference intervals, the values being directly transferable from (commutable with) the established HiCN procedure.

Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement procedure.

BACKGROUND: Monitoring of hemoglobin A(1c) (HbA(1c)) is important in the management of diabetes. The IFCC reference measurement procedure for HbA(1c) is based on the ratio of glycated to nonglycated N-terminal hexapeptides of the beta-chains of hemoglobin after digestion with Glu-C endoproteinase. We developed a modification of the original reference measurement procedure with HPLC-electrospray ionization/mass spectrometry (ESI/MS). METHOD: We performed chromatographic separation of the hexapeptides using a C12 reversed-phase column and a binary gradient system consisting of a mixture of H(2)O/acetonitrile/formic acid. RESULTS: Using this method, we obtained higher signal intensities and improved system stability compared with the reference measurement procedure. In the range of 3% to 14% HbA(1c), intralaboratory CVs were 0.71% to 1.86%. Deviations from IFCC target values were -0.87 to 1.00 relative %. These values fulfill acceptability criteria for HbA(1c) determination set by the IFCC Working Group on HbA(1c) Standardization. CONCLUSIONS: This procedure for the determination of HbA(1c) improves the existing reference measurement procedure.

The IFCC Reference Measurement System for HbA1c: a 6-year progress report.

BACKGROUND: The IFCC Reference Measurement System for hemoglobin (Hb)A(1c) (IFCC-RM) has been developed within the framework of metrologic traceability and is embedded in a network of 14 reference laboratories. This paper describes the outcome of 12 intercomparison studies (periodic evaluations to control essential elements of the IFCC-RM). METHODS: Each study included: unknown samples (to test individual network laboratories); known samples (controls); recently manufactured calibrators (to check calculated assigned value); stored calibrators (to test stability) and a calibration-set (to calibrate the IFCC-RM). The unknown samples are measured by use of the IFCC-RM and the designated comparison methods [DCMs; the National Glycohemoglobin Standardization Program (NGSP) in the US, Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC) in Japan, and Mono-S in Sweden] are used to investigate the stability of the Master Equation (ME), the relationship between IFCC-RM and DCMs. RESULTS: A total of 105 IFCC-RM data sets were evaluated: 95 were approved, 5 were not, and for 5 no data were submitted. Trend analysis of the MEs, expressed as change in percentage HbA(1c) per year, revealed 0.000% (NGSP, not significant), -0.030%, (JDS/JSCC; significant) and -0.016% (Mono-S; not significant). Evaluation of long-term performance revealed no systematic change over time; 2 laboratories showed significant bias, 1 poor reproducibility. The mean HbA(1c) determined by laboratories performing mass spectrometry (MS) was the same as the mean determined by laboratories using capillary electrophoresis (CE), but the reproducibility at laboratories using CE was better. One batch of new calibrators was not approved. All stored calibrators were stable. CONCLUSION: A sound reference system is in place to ensure continuity and stability of the analytical anchor for HbA(1c).

Global standardization of glycated hemoglobin measurement: the position of the IFCC Working Group.

The measurement of glycated hemoglobin is central in the monitoring of glycemic control in patients with diabetes. There are at least 30 different laboratory assays commercially available to measure the proportion of HbA1c in blood. In 1995 the IFCC established a Working Group (IFCC WG-HbA1c) to achieve international standardization of HbA1c measurement. The main achievements can be summarized as follows: a) a reference measurement procedure has been established with purified primary calibrators; b) a network of reference laboratories has been developed worldwide; and c) work has begun on implementation of traceability to the IFCC reference system. The IFCC WG-HbA1c recognizes the recommendation of the IFCC-IUPAC Committee on Nomenclature, Properties and Units that the analyte measured by the IFCC reference measurement procedure has been defined as betaN1-deoxyfructosyl-hemoglobin and that the recommended measurement units are mmol/mol. The IFCC WG-HbA1c recommends maintaining the use of the name HbA1c in clinical practice.

External quality assessment schemes in coagulation in Germany: between regulatory bodies and patient outcome.

In the early 1980s, the first external quality assessment schemes (EQAS) regarding parameters of the coagulation laboratory were established in the daily routine of German laboratories. At present, the EQAS performed by INSTAND offers a wide range of global and single parameters of thrombosis and hemostasis. Only the participation in prothrombin time and activated partial thromboplastin time exercises is mandatory. However, devices applied for patient self-management of oral anticoagulation are not included. This article provides an overview of the EQA activities of INSTAND regarding different coagulation parameters. Problems in acquiring adequate sample material and strategies in establishing acceptability ranges are also discussed.

A novel LC-IDMS/MS method for the determination of the cardiac glycosides digoxin and digitoxin using caesium adducts.

This article describes an essential improvement of the published candidate reference measurement procedure for digoxin and digitoxin and compares it with the original method. The novelty of the method lies in the measurement of the caesium (Cs+) ion as product ion in the multiple reaction monitoring mode (MRM) with potentially improved analytical specificity whilst retaining a comparable accuracy and precision at therapeutic levels. The original measurement procedure used the single-ion mode (SIM). The dissociation of the Cs+ adducts in MRM leads to the formation of Cs+ ions as main charged product in high yield. The present method results in a product ion signal intensity in MRM for digoxin and digitoxin of up to 80% of the precursor ion signal intensity in SIM. The precision, expressed as the coefficient of variation of the new method for digoxin was 3.18% (SIM) and 2.28% (MRM) at a concentration of 0.66 microg/l and 1.26% (SIM) or 1.65% (MRM) at 2.0 microg/l. The corresponding data for digitoxin were 1.21% (SIM) and 1.62% (MRM) at 24 microg/l and 1.46% (SIM) and 1.13% (MRM) at 42 microg/l.

A new approach for the determination of immunosuppressive drugs using HPLC-MS/MS and Cs+ adducts.

In this study a new principle of measurement in LC-MS/MS (liquid chromatography mass spectrometry) for determination of the immunosuppressive drugs sirolimus, everolimus, tacrolimus, and cyclosporin A has been introduced by using the Cs(+) ion as the product ion in the multiple reaction monitoring mode (MRM). Separation of the immunosuppressive agents was achieved using a phenyl-hexyl-RP column together with a ternary gradient elution profile, consisting of water, methanol and acetonitrile combined with 0.1% v/v formic acid and 0.1 mmol/l Cs+. Quantification was performed using cyclosporin D, ascomycin and 32-desmethoxy-rapamycin as internal standards. The inter-run precision of this new method, expressed as the coefficient of variation, was 2.57% for sirolimus, 2.11% for everolimus, 2.31% for tacrolimus and 2.11% for cyclosporin A.

Modification of the IFCC reference measurement procedure for determination of HbA 1c by HPLC-ESI-MS.

The reference measurement procedure for determination of HbA(1c) (glycated haemoglobin) using HPLC(high performance liquid chromatography)-ESI(electrospray ionisation)-MS(mass spectrometry) has been modified. Main modifications were a change in the buffer composition of the HPLC, a change in the gradient elution profile and the introduction of a post-column splitting system. The long-term stability of the HPLC-ESI-MS system proved to be of high importance to get reproducible results. Using the modified HPLC conditions stable retention times, high peak symmetry and a good column batch-to-batch reproducibility of the analytical results were obtained. The mean deviation from the target values was less than 0.7% with an imprecision of less than 2%.

The performance of quantitative D-dimer assays in laboratory routine.

Assessment of D-dimer in plasma is routinely used for the exclusion of venous thrombosis and the monitoring of hypercoagulability. Little information is available about the performance of D-dimer assays in clinical laboratories examined by external quality assessment schemes. We obtained results from 423 laboratories measuring plasma pools from patients with elevated D-dimer levels mixed with human normal plasma. The results from five samples were reported containing D-dimer from the lower normal range up to a 20-fold increased level. In addition, information about the assignment of a cut-off point and the medical need to apply these assays was obtained by standardized questionnaire. Participants reported results and additional information from 20 different assays. Lack of standardization regarding the calibration concepts obstructs comparability of results. Results in one sample varied up to 20-fold between the assays applied. In addition, a high variability was reported around the cut-off values introduced for the exclusion of venous thrombosis and pulmonary embolism. As a consequence, generally accepted cut-off values cannot be established. For cut-off assignment, 62% of participants used the kit insert but also 14% used local validation. In conclusion, standardization or at least harmonization of D-dimer assays is necessary to ensure comparability of D-dimer plasma levels measured in clinical routine.

External quality assessment of tumour marker analysis: state of the art and consequences for estimating diagnostic sensitivity and specificity.

This review shows the current analytical quality for the following analytes used as tumour markers in the external quality assessment (EQA)-programmes of Instand e.V., a national EQA-organiser in Germany: Corticotropin (ACTH), growth hormone (GH, hGH), prolactin (PRL), chorionic gonadotropin (CG, hCG), calcitonin (CT, hCT), thyroglobulin (Tg), carcinoembryonic antigen (CEA), CA-Antigens 125, 72-4, 15-3 and 19-9, alpha foetoprotein (AFP) and prostate-specific antigen (PSA). The results from the participants show a large variation in the precision of the methods used as well as in the comparability of results between methods for the same analyte. In general, the hormones used as tumour markers show better performance than the "CA-markers", which are often inadequately standardised and defined. In the case of one CA-marker (CA 72-4/TAG 72-4), the differences between the lowest kit median concentration and highest kit median concentration for one sample pair were 440% and 580%. The corresponding figures for ACTH were 123% and 156% and for CEA 180% and 184%. The classical tumour markers such as carcinoembryonic antigen (CEA) and alpha foetoprotein (AFP) performed markedly better than the CA-markers and PSA with regards to both inter- and intra-method comparability. The inter-laboratory precision for a given kit and marker was acceptable in many cases. The results show that only results from the same kit/method for each tumour marker can be used for cumulative or time-dependent comparison of results - for example pre-operative and post-operative follow up. In the case of prostate specific antigen (PSA), the kits used for free and total PSA must come from the same producer, if the generally accepted ratios are to have any diagnostic value. The need for kit- and laboratory-specific reference ranges and cut-off values for setting diagnostic specificity and sensitivity is highlighted from the EQA-results. The situation for inter-method comparability for the CA-Markers has not improved over the past decade. With the exception of calcitonin for detecting medullary thyroid carcinoma, chorionic gonadotropin in germ-cell tumours in men and thyroglobulin after total thyroidectomy, none of the remaining analytes appear to be suitable for screening purposes.

Comparability of method results and performance in a national external quality assessment scheme between 1993 and 2003 using thyroid associated analytes as examples.

Six thyroid analytes (free and total triiodothyronine and thyroxine, thyrotropin and thyroglobulin) have been followed up over a 10 year period in a national external quality assessment scheme (EQAS) organised by the Institute for Standardisation and Documentation in the Medical Laboratory (INSTAND). I. The following points were observed: II. The introduction of samples with properties similar to patient serum (filtered, recalcified defibrinated plasma without stripping) improved performance and inter-method comparability for the free thyroid hormones. III. In general, the performance in EQAS has improved over the past decade, an exception being thyroglobulin, where precision has improved at the expense of inter-method comparability. IV. Regular statistical analysis of EQAS data allows adjustment of target ranges to be made when necessary. V. Analytes which are not dependent on binding proteins--thyrotropin and the total thyroid hormones--give rise to similar performance when stripped and spiked plasma or recalcified non-stripped and spiked plasma is used as sample. VI. Whereas certain analytes have had a relatively constant number of participants over the past decade (total thyroid hormones), others have shown a drastic increase (free thyroxine from 67 to 620; thyrotropin from 295 to 724) reflecting the medical demand for the analytes.

Determination of the cardiac glycosides digoxin and digitoxin by liquid chromatography combined with isotope-dilution mass spectrometry (LC-IDMS)--a candidate reference measurement procedure.

This article describes a method of high analytical sensitivity, reproducibility and trueness for the determination of digoxin and digitoxin in serum or plasma at therapeutic levels using a combination of high-pressure liquid chromatography (HPLC), isotope-dilution mass spectrometry (IDMS) and caesium-adduct formation. A method for threefold deuterium substitution in the glycosides was developed, which could be performed within 24 hours without distillation giving yields > 98% of the theoretical value. Extraction from a serum or plasma matrix was performed using a liquid-phase extraction with ammonium acetate buffer/tertiary butylmethyl ether/ethyl acetate at pH 9.5. The HPLC-separation used a 10 x 2 mm LiChrospher RP-18 5 microm guard column in combination with a 125 x 2 mm main column of the same material and a gradient containing methanol, caesium ions and formic acid. Quantification of digoxin and digitoxin was made with IDMS using deuterated internal standards and the system run in single ion monitoring (SIM) mode. The methods had a lower limit of determination of 0.25 microg/l for digoxin and digitoxin, a trueness between 97.5 and 104% for digoxin and between 98 and 101% for digitoxin, respectively and had a coefficient of variation of less than 3% in the therapeutic range for both glycosides. Maximally 1 ml serum or plasma was needed for the procedure. The method is used to set target values for materials used in external quality assessment surveys (EQAS) run by INSTAND as part of a national EQAS-programme.)

Experience with an external quality assessment programme for point-of-care-testing (POCT) devices for the determination of blood glucose.

This article describes the preparation and internal and external evaluation of materials, critical issues in the external quality assessment (EQA) of point-of-care testing (POCT) devices for measuring blood glucose. A comparison was made between different materials, both of natural and synthetic origin and with and without stabilisers. The aims were to produce a material which was compatible with as many POCT-devices as possible and so reduce the number of materials sent out in each campaign as well as to optimise the precision and comparability of results between methods and devices. Although the use of near natural material--sterile-filtered plasma spiked with glucose--survived internal testing, this material proved to be unsuitable for EQA surveys. The study resulted in the reduction of materials for each survey to stabilised whole blood for one device, stabilised plasma for two devices and a synthetic material based on a polyethylene glycol matrix for all other devices. Samples were sent as pairs six times annually. The POCT-devices tested measured precisely but inaccurately in the synthetic material, when compared with the reference method (gas-chromatography coupled with isotope-dilution mass-spectrometry; GC-IDMS), so that the devices could only be evaluated for precision. The construction of ratios between the concentrations measured on the two samples distributed allowed an indirect assessment of accuracy. The need for surveillance of POCT devices is stressed in this publication, which combines theory and practice in setting up and running an EQA programme for blood glucose.