Andrea Mosca

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

Abstract HbA1c is the stable glucose adduct to the N-terminal group of the β-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 β-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.

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).

The role of haemoglobin A2 testing in the diagnosis of thalassaemias and related haemoglobinopathies

The increase in haemoglobin (Hb)A2 level is the most significant parameter in the identification of β thalassaemia carriers. However, in some cases the level of HbA2 is not typically elevated and some difficulties may arise in making the diagnosis. For these reasons the quantification of HbA2 has to be performed with great accuracy and the results must be interpreted together with other haematological and biochemical evidence. The present document includes comments on the need for accuracy and standardisation, and on the interpretation of the HbA2 value, reviewing the most crucial aspects related to this test. A practical flow-chart is presented to summarise the significance of HbA2 estimation in different thalassaemia syndromes and related haemoglobinopathies.

Recommendations for detection and management of unsuitable samples in clinical laboratories

Abstract A large body of evidence attests that quality programs developed around the analytical phase of the total testing process would only produce limited improvements, since the large majority of errors encountered in clinical laboratories still prevails within extra-analytical areas of testing, especially in manually intensive preanalytical processes. Most preanalytical errors result from system flaws and insufficient audit of the operators involved in specimen collection and handling responsibilities, leading to an unacceptable number of unsuitable specimens due to misidentification, in vitro hemolysis, clotting, inappropriate volume, wrong container or contamination from infusive routes. Detection and management of unsuitable samples are necessary to overcome this variability. The present document, issued by the Italian Inter-society SIBioC-SIMeL-CISMEL (Society of Clinical Biochemistry and Clinical Molecular Biology-Italian Society of Laboratory Medicine-Italian Committee for Standardization of Hematological and Laboratory Methods) Study Group on Extra-analytical Variability, reviews the major causes of unsuitable specimens in clinical laboratories, providing consensus recommendations for detection and management. Clin Chem Lab Med 2007;45:728–36.

A review of the challenge in measuring hemoglobin A1c.

The attraction of the simple biochemical concept combined with a clinical requirement for a long-term marker of glycolic control in diabetes has made hemoglobin A1c (HbA1c) one of the most important assays undertaken in the medical laboratory. The diversity in the biochemistry of glycation, clinical requirements, and management demands has resulted in a broad range of methods being developed since HbA1c was described in the late 1960s. A range of analytic principles are used for the measurement of HbA1c. The charge difference between hemoglobin A0 and HbA1c has been widely utilized to separate these two fractions, most notably found these days in ion-exchange high-performance liquid chromatography systems; the difference in molecular structure (affinity chromatography and immunochemical methods) are becoming widely available. Different results found in different laboratories using a variety of HbA1c analyses resulted in the need for standardization, most notably in the United States, Japan, and Sweden. Designated comparison methods are now located in these three countries, but as they are arbitrarily chosen and have differences in specificity, results of these methods and the reference values and action limits of the methods differ and only harmonized HbA1c in specific geographic areas. A reference measurement system within the concept of metrological traceability is now globally accepted as the only valid analytic anchor. However, there is still discussion over the units to be reported. The consensus statement of the International Federation of Clinical Chemistry (IFCC), the American Diabetes Association, the International Diabetes Federation, and the European Association for the Study of Diabetes suggests reporting HbA1c in IFCC units (mmol/mol), National Glycohemoglobin Standardization Program units (%), and estimated average glucose (either in mg/dl or mmol/liter). The implementation of this consensus statement raised new questions, to be answered in a concerted action of clinicians, biochemists, external quality assessment organizers, patient groups, and manufacturers.

The Analytical Goals for Hemoglobin A1c Measurement in IFCC Units and National Glycohemoglobin Standardization Program Units Are Different

To the Editor: The variation of a biological measurand can be expressed in the units of the measured concentrations or as a percentage of the absolute variation relative to the mean concentration. For example, given that different metrologic systems are in use for the measurement of human body temperature, this parameter can be expressed in degrees Celsius (Europe), degrees Fahrenheit (US), or degrees Kelvin (scientists). The equivalent unitary variation is 1.0 °C, 1.8 °F, or 1.0 K, respectively. Expressed as a percentage of the mean body temperature, this variation corresponds to 2.7% (1/37 × 100) for degrees Celsius, 1.8% (1.8/99 × 100) for degrees Fahrenheit, and 0.3% (1/310 × 100) for degrees Kelvin. From these results, one might conclude that temperature variation is lowest for scientists and highest for Europeans. Of course, that is nonsense. This wrong conclusion derives from the fact that variation across metrologic systems cannot merely be compared in terms of relative percentages when the y intercept (b) in the generic conversion equation ( y = a x + b) is not equal to zero. A higher y -intercept value will have a …

Revaluation of biological variation of glycated hemoglobin (HbA1c) using an accurately designed protocol and an assay traceable to the IFCC reference system

BACKGROUND Glycated hemoglobin (HbA(1c)) has a key role for diagnosing diabetes and monitoring glycemic state. As recently reviewed, available data on HbA(1c) biological variation show marked heterogeneity. Here we experimentally revaluated these data using a well designed protocol. METHODS We took five EDTA whole blood specimens from 18 apparently healthy subjects on the same day, every two weeks for two months. Samples were stored at -80°C until analysis and assayed in duplicate in a single run by Roche Tina-quant® Gen.2 immunoassay. Data were analyzed by the ANOVA. To assess the assay traceability to the IFCC reference method, we preliminarily carried out a correlation experiment. RESULTS The bias (mean±SD) of the Roche immunoassay was 0.3%±0.7%, confirming the traceability of the employed assay. No difference was found in HbA(1c) values between men and women. Within- and between-subject CV were 2.5% and 7.1%, respectively. Derived desirable analytical goals for imprecision, bias, and total error resulted 1.3%, 1.9%, and 3.9%, respectively. HbA(1c) had marked individuality, limiting the use of population-based reference limits for test interpretation. The estimated critical difference was ~10%. CONCLUSIONS For the first time we defined biological variation and derived indices for the clinical application of HbA(1c) measurements using an accurately designed protocol and an assay standardized according to the IFCC.

Biological variability of glycated hemoglobin.

BACKGROUND The measurement of glycated hemoglobin (HbA(1c)) has a pivotal role in monitoring glycemic state in diabetic patients. Furthermore, the American Diabetes Association has recently recommended the use of HbA(1c) for diabetes diagnosis, but a clear definition of the clinically allowable measurement error is still lacking. Information on biological variability of the analyte can be used to achieve this goal. METHODS We systematically reviewed the published studies on the biological variation of HbA(1c) to check consistency of available data in order to accurately define analytical goals. RESULTS The nine recruited studies were limited by choice of analytic methodology, population selection, protocol application and statistical analyses. CONCLUSIONS There is an urgent need to determine biological variability of HbA(1c) using a specific and traceable assay, appropriate protocol and appropriate statistical evaluation of data.

New analytical tools and epidemiological data for the identification of HbA2 borderline subjects in the screening for beta-thalassemia

The increase of HbA(2) is the most important feature in the identification of beta-thalassemia carriers. However, some carriers are difficult to identify, because the level of HbA(2) is not in the typical range. Few data are available concerning the prevalence of such unusual phenotypes, and knowing their expected prevalence could be helpful in detecting systematic drifts in the analytical systems for HbA(2) quantification. In this study we report a retrospective investigation in two centres with high prevalence of beta-thalassemia. The prevalence of borderline subjects was found to be 2.2 and 3.0%, respectively. The genotypes of a subgroup of these subjects were then analyzed and in about 25% of cases a mutation in the globin genes was identified. We conclude that the occurrence of HbA(2) borderline phenotypes is not a rare event. In order to obtain more accurate HbA(2) measurements the development of an international reference measurement system for HbA(2), based on quantitative peptide mapping, has been recently started. We believe that the innovative approach of our method could also be used as a model to develop accurate quantitative methods for other red cell proteins relevant to the biodynamic properties and the surface electrochemistry of erythrocytes.

Glycemic control in the clinical management of diabetic patients.

Abstract In clinical practice, glycemic control is generally assessed by measuring and interpreting glycated hemoglobin levels, however, this test should be run under standardized conditions. We focus here on the crucial steps to ensure IFCC standardized HbA1c results, pointing out several residual weak points, mostly relating to the laboratory end-user (calibration, quality control materials, and EQAS). We also review the use of HbA1c for diagnosing diabetes and the various indicators useful for assessing glucose variability because in some cases they seem to represent a patient’s glucose profile more accurately than one-off HbA1c assays. Finally, the potential utility of glycated albumin and the glycation gap, the costs involved and the laboratory management issues are briefly discussed.