Renata Paleari

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.

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.

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.

The relevance of hemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies.

OBJECTIVES The increase in hemoglobin (Hb) F level is variably associated to the presence of beta thalassemia trait, and is more typical in presence of deltabeta thalassemia and of hereditary persistence of fetal hemoglobin. In normal healthy subjects variable levels of HbF are related to the presence of the polymorphism (G)gamma -158 (C>T). Moreover, HbF can also be variably increased in association with other acquired conditions. The objective of this work is to review the role of the determination of HbF in various conditions. DESIGN AND METHODS In the present document we comment on the need for accuracy and standardization, and on the interpretation of the HbF value, reviewing most crucial aspects related to this test. RESULTS We present a practical flow-chart summarizing the significance of the HbF estimation in different thalassemia syndromes and related hemoglobinopathies. CONCLUSION The determination of HbF is relevant for the final diagnosis of various physiopathological conditions. In our opinion its importance will increase in the following years, because of the proliferation of novel approaches for the induction of HbF synthesis as a cure for thalassemia syndromes.

Biological Variability of Albumin Excretion Rate and Albumin-to- Creatinine Ratio in Hypertensive Type 2 Diabetic Patients

Abstract The importance of measuring microalbuminuria is well established. However, only scanty data are available concerning the biological variability of albumin excretion in type 2 diabetic subjects. We report our experience from a large clinical trial of a new antihypertensive drug (Lercanidipine) designed to reduce albumin excretion and blood pressure in type 2 diabetic patients with hypertension and microalbuminuria. Eighty seven patients with persistent microalbuminuria were studied within 1 year of the clinical trial. The measurements were performed on blood and timed urine samples frozen at −80 °C and shipped to a central laboratory unit. Preliminary experiments were performed to assess albumin stability in urine under various conditions (4 °C, −20 °C and −80 °C), particularly with regard to the albumin/creatinine ratio. Urine samples can be stored up to 3 weeks at 4 °C or up to 2 months at −80 °C. The biological variability of the albumin excretion rate was 25.7%, while that of the albumin/creatinine ratio was 13.4%. These data are useful in defining the analytical goals of imprecision for microalbuminuria (CV = 13% for albumin, and CV = 6% for albumin/creatinine ratio). No correlation between albumin/creatinine ratio and HbA1c was found in the cohort of 61 microalbuminuric patients who completed the trial. The results of this study confirm that the albumin/creatinine ratio is much more suitable for monitoring albumin excretion in longitudinal studies than the albumin excretion rate.

Evaluation of biological variation of glycated albumin (GA) and fructosamine in healthy subjects

BACKGROUND Glycated albumin (GA) and fructosamine are nonenzymatically glycated proteins still frequently utilized for monitoring glycemic control in diabetics. To investigate the analytical variation and the degree of individuality of these glycemic markers, we have performed an experimental study under a well designed and standardized protocol. METHODS We collected five specimens from each of 18 apparently healthy subjects (9 men and 9 women, ages 26-52 years), 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 analytical run. GA and fructosamine were measured using enzymatic (Lucica®GA-L, Asahi Kasei Pharma, AKP, Tokyo, Japan) and colorimetric assays, respectively, on a Modular P Roche system (Roche Diagnostics GmbH, Mannheim, Germany). Data were analyzed by ANOVA. RESULTS Analytical coefficient of variation (CVA) was 1.7%, 2.3% and 2.8% for GA, albumin and fructosamine, respectively. Within-subject (CVW) and between-subject (CVG) coefficients of variation were 2.1% and 10.6% for GA, 2.3% and 2.9% for albumin, and 2.3% and 6.3% for fructosamine. The estimated critical difference (CD) was 7.5% for GA, 9% for albumin and 10% for fructosamine. CONCLUSIONS The good quality achieved by the analytical method for GA assessment and the reduced within-subject biological variation would allow to recommend this test in clinical practice for evaluation of glycemic control along with measurement of glycated hemoglobin.

External quality assessment of hemoglobin A2 measurement: data from an Italian pilot study with fresh whole blood samples and commercial HPLC systems

Abstract Background: To evaluate the extent of interlaboratory variation and accuracy in hemoglobin A2 (HbA2) assays, a pilot study of external quality assessment was organized among 48 Italian laboratories routinely measuring HbA2. As part of the study, a survey was also performed by sending a questionnaire concerning some important analytical aspects related to the determination of HbA2. Methods: The trial specimens consisted of three whole blood samples (A, B and C) with normal, pathological and borderline HbA2 content, respectively. All laboratories used HPLC analyzers from the same manufacturer (Bio-Rad Laboratories). Results: Normal and pathological samples were clearly differentiated by all laboratories, while data for the borderline sample partially overlapped those for the other samples. The overall interlaboratory coefficient of variation was 8.0%, 6.0% and 7.9% for samples with low, high and intermediate HbA2 levels, respectively. To assign HbA2 target values to the samples, the median of the laboratory group was used. The accuracy of HbA2 results was evaluated on the basis of allowable total error. The proportion of laboratories reporting unacceptable results was 31.9% (15 out of 47) for sample A, 17.0% (8 out of 47) for sample B, and 31.9% (15 out of 47) for sample C. No abnormalities in the chromatographic separation pattern were reported by any of the laboratories. Conclusions: We conclude that quality in the measurement of HbA2 should be improved. Clin Chem Lab Med 2007;45:88–92.

Evaluation of the analytic performances of the new HPLC system HLC-723 G7 for the measurement of hemoglobin A1c.

OBJECTIVES The analytical performance of a new automated HPLC system, for the determination of HbA1C in blood (Tosoh HLC-723 G7), was studied. DESIGN AND METHODS The study design included the evaluation of imprecision, linearity, interference and carryover. Comparison study was performed by comparing HbA1C results with those obtained from an established method (Bio-Rad Variant II). RESULTS Total imprecision was less than 1.34% and the results were linear up to 17.2% HbA1C. The method showed a wide analytical range, and no carryover between specimens. Comparison study yielded, r=0.989, Sy.x=0.255, regression equation (y=0.9895x-0.35); Bland-Altman plot showed a mean bias=- 0.43% HbA1C with confidence limits ranging from -0.48% to -0.38% HbA1C. The presence of abnormal hemoglobin was clearly revealed, and no interference from labile HbA1C was apparent. CONCLUSION The HLC-723 G7 instrument seems to be a reliable system for routine assay of HbA1C.

Performance of glycated hemoglobin (HbA1c) methods evaluated with EQAS studies using fresh blood samples: Still space for improvements

BACKGROUND The determination of glycated hemoglobin is a key indicator for the management of diabetic patients. A reference measurement system for its determination is available and IVD manufacturers should have aligned their assay to this system. METHODS Two fresh blood samples were distributed by courier to 206 Italian laboratories asking for the determination of their HbA1c concentration. Target HbA1c values were assigned by the IFCC reference measurement procedure. RESULTS From 193 laboratories using analytical systems from five manufacturers (Bio-Rad Laboratories, A. Menarini Diagnostics, Roche Diagnostics, Sebia and Tosoh), we obtained a global variability of 5.3% (in terms of CV) and of 3.8% at an HbA1c value of 37.4 mmol/mol (sample 1) and 62.0 mmol/mol (sample 2), respectively. With a goal for the allowable total error (TE) of 6.0%, 70% and 77% of the participants met this criterion for samples 1 and 2, respectively. Inter-laboratory CVs, were between 3.3 and 5.0% and between 2.2 and 3.7% for samples 1 and 2, respectively. Tosoh users registered the smallest inter-laboratory CV in sample 1, and Sebias in sample 2. With regard to trueness, all methods had a mean bias of ≤ 2.8% with respect to the target values, with the exception of Tosoh (bias of + 6.1 and + 5.8%, for samples 1 and 2, respectively). CONCLUSION These results are in good agreement with those obtained by the CAP 2014 GH2-A survey, suggesting then that still there is an urgent need for improving a significant part of the methods currently used to measure HbA1c.

Genetic variability of the fructosamine 3-kinase gene in diabetic patients.

Abstract Background: Nonenzymatic glycation appears to be an important factor in the pathogenesis of diabetic complications. Fructosamine 3-kinase (FN3K), initially identified in erythrocytes, appears to be responsible for the removal of fructosamine from proteins, suggesting a protective role in nonenzymatic glycation. Recently, genetic variants in the FN3K gene have been studied in diabetic patients. The aim of our study was the molecular characterization of the FN3K gene in a representative group of Italian patients with type 1 (T1DM) and 2 (T2DM) diabetes mellitus and in a cohort of healthy controls. Methods: Seventy diabetic subjects (35 type 1 and 35 type 2) with stable glycemic control and 33 healthy control subjects were evaluated using PCR and direct sequencing of the FN3K gene. Denaturing high performance liquid chromatography (DHPLC) was used in controls for screening for the presence of the genetic variants previously found in diabetic patients. Results: Seven different genetic variants were identified, five of them already reported and two new: the p.R187X and p.Y239C mutations identified in two females affected by T2DM. No significant association was found between certain polymorphisms and diabetes conditions. Preliminary haplotype studies are also reported. With respect to genotypes, we noted that some were not present in all the investigated cohort, and some were found related to higher glycated hemoglobin compared to others, although not at a significant level, probably because of the small number of subjects investigated. Conclusions: In conclusion, this study identified two new mutations and additional variants within the FN3K gene. This is the first study on FN3K in Italy. Future work is needed to achieve a better understanding of the FN3K enzyme and its possible clinical utility in the management of diabetic patients.