Harmonization of measurement results of the alcohol biomarker carbohydrate-deficient transferrin by use of the toolbox of technical procedures of the International Consortium for Harmonization of Clinical Laboratory Results.

BACKGROUND: The need for equivalent results of routine measurement procedures for the alcohol biomarker carbohydrate-deficient transferrin (CDT) has been recognized by the IFCC. This article describes a project to harmonize CDT as conducted by an IFCC working group initiated for this purpose. METHODS: We used procedures for achieving harmonization as developed by the Consortium for Harmonization of Clinical Laboratory Results to assess the suitability of a candidate reference measurement procedure (cRMP), candidate reference materials (cRMs), and the success of efforts to achieve harmonization. RESULTS: CDT measurement procedures in routine use showed good reproducibility (CV 1.1%-2.8%) and linearity (r > 0.990) with variable slopes (0.766-1.065) and intercepts (-0.34 to 0.92) compared to the cRMP. Heterogeneity after simulated harmonization was 4.7%. cRMs of frozen human native sera demonstrated commutability and 3-year stability for routine measurement procedures. The cRMP provided reproducible value assignment to cRMs with an expanded uncertainty (k = 2) of 0.03% at the 1.2% CDT level and 0.06% at the 4.4% CDT level. Harmonization efforts reduced the intermeasurement CV from 8.8% to 3.4%, allowed 99% recovery of the values assigned with the cRMP, and demonstrated 99% of results within the desirable allowable total error. Harmonization was less successful in samples with low CDT and high trisialotransferrin concentrations. CONCLUSIONS: Harmonization of CDT is possible with frozen human native sera as cRMs with values assigned by use of the cRMP. We propose the cRMP as a candidate international conventional reference measurement procedure and cRMs as candidate international calibrators.

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: III. Performance of native serum and serum spiked with disialotransferrin proves that harmonization of CDT assays is possible.

Carbohydrate-deficient transferrin (CDT) is a generic term that refers to the transferrin glycoforms whose concentration in blood is temporarily increased by sustained alcohol consumption. Due to high clinical specificity, CDT was proposed as a biomarker of heavy alcohol use and has been available for about 20 years. A number of methods have been developed for CDT measurement based on different analytical techniques and principles and without any harmonization or calibration to a reference method. As a consequence, neither the reference limits nor the cut-off values have been similar across assays, hampering understanding of the diagnostic value of CDT and its routine use. This prompted the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to initiate a Working Group on Standardization of CDT (WG-CDT). This third publication of the WG-CDT is devoted to testing the commutability of native and disialotransferrin-spiked serum panels as candidate secondary reference materials, in order to prove the harmonization potential of commercial CDT methods. The results showed that assay harmonization reduced the inter-laboratory imprecision in a network of reference laboratories running the HPLC candidate reference method. In the seven commercial methods evaluated in this study, the use of multi-level secondary calibrators of human serum origin significantly reduced the between-method imprecision. Thus, harmonization of CDT measurements by different methods can be achieved using this calibration system, opening the way for a full standardization of commercial methods against a reference method by use of certified reference materials.

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: II. Performance of a laboratory network running the HPLC candidate reference measurement procedure and evaluation of a candidate reference material.

Carbohydrate-deficient transferrin (CDT) is a descriptive term used for a temporary change in the transferrin glycosylation profile caused by alcohol, and used as a biomarker of chronic high alcohol consumption. The use of an array of methods for measurement of CDT in various absolute or relative amounts, and sometimes covering different transferrin glycoforms, has complicated the comparability of results and caused confusion among medical staff. This situation prompted initiation of an IFCC Working Group on CDT standardization. This second publication of the WG-CDT covers the establishment of a network of reference laboratories running a high-performance liquid chromatography (HPLC) candidate reference measurement procedure, and evaluation of candidate secondary reference materials. The network laboratories demonstrated good and reproducible performance and thus can be used to assign target values for calibrators and controls. A candidate secondary reference material based on native human serum lyophilized with a cryo-/lyoprotectant to prevent protein denaturation was found to be commutable and stable during storage. A proposed strategy for calibration of different CDT methods is also presented. In an external quality assurance study involving 66 laboratories and covering the current routine CDT assays (HPLC, capillary electrophoresis and immunoassay), recalculation of observed results based on the nominal values for the candidate calibrator reduced the overall coefficient of variation from 18.9% to 5.5%. The logistics for distribution of reference materials and review of results were found to be functional, indicating that a full reference system for CDT may soon be available.

Statistical methods for monitoring the relationship between the IFCC reference measurement procedure for hemoglobin A1c and the designated comparison methods in the United States, Japan, and Sweden.

BACKGROUND: The American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD)/International Diabetes Federation (IDF)/IFCC Consensus Statement on the worldwide standardization of HbA(1c) states that "... [HbA(1c)] results are to be reported world-wide in IFCC units ... and derived NGSP units ... , using the IFCC-NGSP master equation." METHODS: We describe statistical methods to evaluate and monitor the relationships as expressed in master equations (MEs) between the IFCC Reference Measurement procedure (IFCC-RM) and designated comparison methods (DCMs) [US National Glycohemoglobin Standardization Program (NGSP), Japanese Diabetes Society/Japanese Society for Clinical Chemistry (JDS/JSCC), and Mono-S in Sweden]. We applied these statistics, including uncertainty calculations, to 12 studies in which networks of reference laboratories participated, operating the IFCC-RM and DCMs. RESULTS: For NGSP and Mono-S, slope, intercept, and derived percentage HbA(1c) at the therapeutic target show compliance with the respective MEs in all 12 studies. For JDS/JSCC, a slight deviation is seen in slope and derived percentage HbA(1c) in 2 of the 12 studies. Using the MEs, the uncertainty in an assigned value increases from 0.42 mmol/mol HbA(1c) (IFCC-RM) to 0.47 (NGSP), 0.49 (JDS/JSCC), and 0.51 (Mono-S). CONCLUSIONS: We describe sound statistical methods for the investigation of relations between networks of reference laboratories. Application of these statistical methods to the relationship between the IFCC-RM and DCMs in the US, Japan, and Sweden shows that they are suitable for the purpose, and the results support the applicability of the ADA/EASD/IDF/IFCC Consensus Statement on HbA1c measurement.

Reprint of Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

IFCC approved HPLC reference measurement procedure for the alcohol consumption biomarker carbohydrate-deficient transferrin (CDT): Its validation and use.

Carbohydrate-deficient transferrin (CDT) is used as a biomarker of sustained high alcohol consumption. The currently available measurement procedures for CDT are based on various analytical techniques (HPLC, capillary electrophoresis, nephelometry), some differing in the definition of the analyte and using different reference intervals and cut-off values. The Working Group on Standardization of CDT (WG-CDT), initiated by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), has validated an HPLC candidate reference measurement procedure (cRMP) for CDT (% disialotransferrin to total transferrin based on peak areas), demonstrating that it is suitable as a reference measurement procedure (RMP) for CDT. Presented is a detailed description of the cRMP and its calibration. Practical aspects on how to treat genetic variant and so-called di-tri bridge samples are described. Results of method performance characteristics, as demanded by ISO 15189 and ISO 15193, are given, as well as the reference interval and measurement uncertainty and how to deal with that in routine use. The correlation of the cRMP with commercial CDT procedures and the performance of the cRMP in a network of laboratories are also presented. The performance of the CDT cRMP in combination with previously developed commutable calibrators allows for standardization of the currently available commercial measurement procedures for CDT. The cRMP has recently been approved by the IFCC and will be from now on be known as the IFCC-RMP for CDT, while CDT results standardized according to this RMP should be indicated as CDTIFCC.

Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin.

Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent. Two samples from Bio-Rad (Lyphochek)--one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)--were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(0), which is major Hb component containing two alpha chains and two beta chains; HbA(1c), which is post-translational glycation on the N-terminus of the beta chains of HbA(0); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(0) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.

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).

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: I. Analyte definition and proposal of a candidate reference method.

An alcohol-associated change in the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker of chronic moderate to heavy alcohol consumption. A current limitation in CDT analysis is the lack of standardization, which hampers clinical and analytical comparison between studies. This situation prompted initiation of a Working Group (WG) on CDT Standardization under the auspices of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The standardization work aims to define and validate the analyte, select a reference method, work out procedures for the production of reference materials, and make suggestions for the clinical usage of CDT. The first recommendation of the WG is that disialotransferrin should be the primary target molecule for CDT measurement and the single analyte on which CDT standardization is based. It is further recommended that HPLC should be the analytical principle considered as the basis of an interim reference method until a suitable mass spectrometric reference method is established. In clinical use, CDT should be expressed in a relative amount (% CDT), to compensate for variations in the total transferrin concentration.

Improved HPLC method for carbohydrate-deficient transferrin in serum.

BACKGROUND: There is need for a reference method for transferrin glycoforms in serum to which routine immunologic methods for the alcohol marker carbohydrate-deficient transferrin (CDT) can be traceable. We describe an improved HPLC method for transferrin glycoforms. METHODS: Transferrin was iron-saturated by mixing the serum with ferric nitrilotriacetic acid, and lipoproteins were precipitated with dextran sulfate and calcium chloride. Separation of glycoforms was performed on a SOURCE 15Q anion-exchange column using salt gradient elution. Quantification relied on selective absorbance of the iron-transferrin complex at 470 nm. The relative amount of each glycoform was calculated as a percentage of the area under the curve, using baseline integration. RESULTS: The HPLC system provided reproducible separation and quantification of the asialo-, monosialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialotransferrin glycoforms. Most importantly, disialo- and trisialotransferrin were almost baseline separated. The intra- and interassay CV for disialotransferrin were <5%. Serum and the pretreated samples were stable for at least 2 days at 22 or 4 degrees C. Sera from 132 healthy controls contained [mean (SD)] 1.16 (0.25)% disialotransferrin, 4.77 (1.36)% trisialotransferrin, 80.18 (2.01)% tetrasialotransferrin, and 13.88 (1.69)% pentasialo- + hexasialotransferrin. In some cases of a high (>6%) trisialotransferrin, monosialotransferrin was detected at <0.25%. Asialotransferrin was not detected in control sera, but was detected in 57% of chronic heavy drinkers and in 62% of sera with >/=2% disialotransferrin. CONCLUSIONS: The HPLC method fulfills the requirements of a preliminary reference method for CDT and should work for any combination of serum transferrin glycoforms. This method could also be useful for confirming positive CDT results by immunoassays in medico-legal cases.

Urinary excretion of total cystine and the dibasic amino acids arginine, lysine and ornithine in relation to genetic findings in patients with cystinuria treated with sulfhydryl compounds.

Advances in molecular genetics have brought a deeper understanding of cystinuria. This autosomal recessive disease, which is caused by a defective tubular reabsorption of cystine and the three dibasic amino acids arginine, lysine and ornithine, results in a lifelong risk of renal stone formation because of the low solubility of cystine in urine. Mutations detected within the two genes known to be associated with cystinuria, SLC3A1 (related to type I) and SLC7A9 (related to non-type I), cannot, however, in all cases explain the disease. Inasmuch as a high urinary concentration of cystine is the basis of stone formation in these patients, our aim was to measure urinary total cystine, arginine, lysine and ornithine, in patients currently lacking a full genetic explanation for their disease. Thirty-three patients with cystinuria who were on long-term treatment with tiopronin or D-penicillamine were divided into two groups. Group 1 comprised eight patients who carried mutation in one of the SLC3A1 alleles and two patients who completely lacked mutations both in the SLC3A1 and the SLC7A9 genes, that is genetic findings discordant with the increased urinary excretion of cystine and the dibasic amino acids in these patients. Group 2 comprised 23 patients homozygous for mutations within SLC3A1, that is genetic findings in accordance with the excretion pattern of classic type I cystinuria. When the two groups were compared, Group 1 had a significantly higher total urinary excretion of cystine ( p<0.01) as well as of arginine, lysine and ornithine ( p<0.05) than Group 2. Also, when the two patients without mutations were excluded from the calculations, there still was a significant difference in the urinary excretion of total cystine ( p<0.05). This suggests that the two patients without any detected mutations in the two known cystine transport genes also contributed to the difference. These unexpected findings indicate that an additional gene or genes participate in the urinary cystine reabsorption in the cystinuric patients who currently are without a full genetic explanation for their disease.

Approved IFCC reference method for the measurement of HbA1c in human blood.

HbA1C is the stable glucose adduct to the N-terminal group of the beta-chain of HbA0. The measurement of HbA1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the beta-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA1c and HbA0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA1c routine assays worldwide.

IFCC reference system for measurement of hemoglobin A1c in human blood and the national standardization schemes in the United States, Japan, and Sweden: a method-comparison study.

BACKGROUND: The national programs for the harmonization of hemoglobin (Hb)A(1c) measurements in the US [National Glycohemoglobin Standardization Program (NGSP)], Japan [Japanese Diabetes Society (JDS)/Japanese Society of Clinical Chemistry (JSCC)], and Sweden are based on different designated comparison methods (DCMs). The future basis for international standardization will be the reference system developed by the IFCC Working Group on HbA(1c) Standardization. The aim of the present study was to determine the relationships between the IFCC Reference Method (RM) and the DCMs. METHODS: Four method-comparison studies were performed in 2001-2003. In each study five to eight pooled blood samples were measured by 11 reference laboratories of the IFCC Network of Reference Laboratories, 9 Secondary Reference Laboratories of the NGSP, 3 reference laboratories of the JDS/JSCC program, and a Swedish reference laboratory. Regression equations were determined for the relationship between the IFCC RM and each of the DCMs. RESULTS: Significant differences were observed between the HbA(1c) results of the IFCC RM and those of the DCMs. Significant differences were also demonstrated between the three DCMs. However, in all cases the relationship of the DCMs with the RM were linear. There were no statistically significant differences between the regression equations calculated for each of the four studies; therefore, the results could be combined. The relationship is described by the following regression equations: NGSP-HbA(1c) = 0.915(IFCC-HbA(1c)) + 2.15% (r(2) = 0.998); JDS/JSCC-HbA(1c) = 0.927(IFCC-HbA(1c)) + 1.73% (r(2) = 0.997); Swedish-HbA(1c) = 0.989(IFCC-HbA(1c)) + 0.88% (r(2) = 0.996). CONCLUSION: There is a firm and reproducible link between the IFCC RM and DCM HbA(1c) values.