Publications by authors named "Renata Paleari"

35 Publications

Why glycated albumin decreases in pregnancy? Evidences from a prospective study on physiological pregnancies of Caucasian women.

Clin Chim Acta 2021 Sep 3;520:217-218. Epub 2021 Jun 3.

Dip. Di Fisiopatologia medico-chirurgica e dei trapianti, Università degli Studi di Milano, Milano, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.cca.2021.05.035DOI Listing
September 2021

Placental Antioxidant Defenses and Autophagy-Related Genes in Maternal Obesity and Gestational Diabetes Mellitus.

Nutrients 2021 Apr 15;13(4). Epub 2021 Apr 15.

Department of Biomedical and Clinical Sciences "Luigi Sacco", Università degli Studi di Milano, 20157 Milano, Italy.

Maternal obesity and gestational diabetes mellitus (GDM) are increasing worldwide, representing risk factors for both mother and child short/long-term outcomes. Oxidative stress, lipotoxicity and altered autophagy have already been reported in obesity, but few studies have focused on obese pregnant women with GDM. Antioxidant and macro/chaperone-mediated autophagy (CMA)-related gene expressions were evaluated herein in obese and GDM placentas. A total of 47 women with singleton pregnancies delivered by elective cesarean section were enrolled: 16 normal weight (NW), 18 obese with no comorbidities (OB GDM(-)), 13 obese with GDM (OB GDM(+)). Placental gene expression was assessed by real-time PCR. Antioxidant gene expression (, , ) decreased, the pro-autophagic gene increased and the chaperone-mediated autophagy regulator decreased in OB GDM(-) vs. NW. On the other hand, expression increased in OB GDM(+) vs. OB GDM(-). When analyzing results in relation to fetal sex, we found sexual dimorphism for both antioxidant and CMA-related gene expressions. These preliminary results can pave the way for further analyses aimed at elucidating the placental autophagy role in metabolic pregnancy disorders and its potential targetability for the treatment of diabetes outcomes.
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http://dx.doi.org/10.3390/nu13041303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071310PMC
April 2021

A roadmap for the standardization of hemoglobin A.

Clin Chim Acta 2021 Jan 10;512:185-190. Epub 2020 Nov 10.

Dept. of Clinical Genetics/LDGA, Leiden University Medical Center, Leiden, the Netherlands.

Background: Standardization of laboratory tests can be a long process, and this is the case with regards to the methods used to measure hemoglobin A (HbA), an important marker for beta-thalassemia and other thalassemic conditions. The IFCC standardization project started in 2004, and it took at least 15 years before developing a reference measurement procedure, defining and producing calibrators and certified reference materials.

Methods: A series of steps have to be undertaken in order to promote the standardization in the field, a process involving a number of stakeholders (manufacturers, scientific societies, national health bodies, laboratory professionals, clinicians). In this work we describe some possible process indicators, in order to assure that the standardization will have internal and external validity and be effective for a long time. These indicators concern the inter-method studies, elaboration of External Quality Assessment Schemes, and the evaluation of the yearly distributions of HbA measurements collected in selected laboratories.

Results: Preliminary results are reported concerning the yearly distributions of HbA, collected in two different locations, and using different analytical methods. Median yearly values were found very constant over the years, but different between methods. On the other side, results obtained on the same specimens using two different techniques, proved that results by capillary electrophoresis in 2 out of the 3 years of observation, were significantly lower than those by HPLC.

Conclusion: In this document we report what has been done so far, and what has to be done to achieve the standardization of the measurement of HbA worldwide.
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http://dx.doi.org/10.1016/j.cca.2020.11.008DOI Listing
January 2021

Role of fructosamine-3-kinase in protecting against the onset of microvascular and macrovascular complications in patients with T2DM.

BMJ Open Diabetes Res Care 2020 05;8(1)

Department of Medicine (DIMED), University of Padova School of Medicine and Surgery, Padova, Italy.

Introduction: Microangiopathic and macroangiopathic complications are the main cause of morbidity and mortality in the diabetic population. Numerous publications have highlighted the role of glycation in the onset of complications of diabetes. In this context, the detection of fructosamine-3-kinase (FN3K)-an enzyme capable of counteracting the effect of hyperglycemia by intervening in protein glycation-has attracted great interest. Several studies have linked genetic variability to its enzymatic activity and glycated hemoglobin (HbA1c) levels. Here, we investigated the role of polymorphisms in the development of microvascular and macrovascular complications of diabetes.

Research Design And Methods: The anthropometric and biochemical parameters, and any medical history of microangiopathic and macroangiopathic complications, were documented in a sample of 80 subjects with type 2 diabetes. All subjects were screened for gene and analyzed for the combination of three polymorphisms known to be associated with its enzymatic activity (rs3859206 and rs2256339 in the promoter region and rs1056534 in exon 6).

Results: The combination of allelic variants of polymorphisms resulted in 13 distinct genotypic variants within the cohort. Comparison between genotypes showed no significant differences in terms of demographic, anthropometric and biochemical parameters, risk markers and long-term complications, except for a higher age and vitamin E levels associated with the genotype presenting GG at position -385, TT at position -232, and CC at c.900 A. Evaluating the microangiopathic and macroangiopathic complications as a whole, we found that they appeared significantly less present in this genotype compared with all other genotypes (=0.0306).

Conclusions: The group of patients carrying the favorable allele for the three polymorphisms of the gene revealed less severe microangiopathy and macroangiopathy, suggesting a protective role of this genotype against the onset of the complications of diabetes.
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http://dx.doi.org/10.1136/bmjdrc-2020-001256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259852PMC
May 2020

The analytical performance of laboratory plasma glucose and HbA measurements are largely acceptable.

Acta Diabetol 2020 Feb 21;57(2):215-219. Epub 2019 Aug 21.

Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Via Fratelli Cervi 93, 20090, Segrate, Milan, Italy.

Aims: Poor comparability between laboratory results may have a strong impact on clinical decisions. The aim of this study was to assess the quality of glucose and HbA measurements in a large cohort of laboratories in various countries, in order to evaluate whether the current state of these very basic laboratory examinations can be considered to be adequate with respect to the clinical needs in the management of glucose control in diabetic patients.

Methods: External quality assessment schemes and proficiency testing surveys performed in 2017 in several European and American laboratories were analyzed in order to estimate the percentage of laboratories reaching the desired quality criteria based on the allowable total error in accordance with various international recommendations.

Results: In 2017 more than 95% of laboratories met the allowable total error for measuring HbA, and 92-94% of the studied laboratories met the target for glucose measurement.

Conclusions: The analytical quality for measuring glycated hemoglobin and glucose at laboratory level is generally acceptable, and accreditation to the ISO 15189:2012 standard is a robust guarantee that the laboratory meets the required criteria of acceptability. Several pre-analytical factors which may explain the discrepancies between the measured HbA and that estimated from other indicators of glucose control have to be taken into account, by focusing more on the pre-analytical than the analytical phase. In the case of glucose, special attention should be paid to the use of the correct anticoagulant, in order to avoid false negative results.
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http://dx.doi.org/10.1007/s00592-019-01408-4DOI Listing
February 2020

Effects of different anticoagulants on glycated albumin quantification.

Biochem Med (Zagreb) 2019 Feb 15;29(1):010901. Epub 2018 Dec 15.

Clinical Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.

Introduction: In the last 20 years glycated albumin (GA) measurement has been demonstrated to be a reliable glycation marker and recently as the most innovative one in western countries. Glycated albumin has been already adopted by some Asian countries due to its usefulness in diabetes screening. The aim of the present study was to investigate for the first time the effects of different anticoagulants on GA assay.

Materials And Methods: From each of 60 patients a serum tube and KEDTA, Li-Heparin and NaF-EDTA containing tubes were collected. All tubes were from Sarstedt (Verona, Italy). Glycated albumin was measured in duplicate in each sample tube in a single analytical run with quantILab glycated albumin (Instrumentation Laboratory SpA - A Werfen Company, Milan, Italy) on Architect c8000 analyser (Abbott SRL, Rome, Italy). Comparison of GA% in evaluated tubes was made by paired Wilcoxon test.

Results: Median and interquartile range GA% concentrations were 15.4% (13.2 - 19.1) in serum, 15.7% (13.6 - 19.9) in KEDTA, 15.6% (13.3 - 19.7) in Li-heparin and 15.5% (13.1 - 19.3) in NaF-EDTA samples, respectively. Glycated albumin mean relative bias respect to serum was within desirable bias derived from biological variation studies (± 2.9%) when KEDTA (+ 2.8%), Li-heparin (+ 0.9%) or NaF-EDTA (+ 0.1%), were used as anticoagulants.

Conclusions: Our results demonstrate that the GA% assay is not affected by relevant interferences when KEDTA, Li-heparin or NaF-EDTA are used as anticoagulants, so they can be used interchangeably without a relevant impact on the clinical use of the test.
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http://dx.doi.org/10.11613/BM.2019.010901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294155PMC
February 2019

Standardization of the HbA Assay.

EJIFCC 2018 Dec 5;29(4):298-302. Epub 2018 Dec 5.

Department of Physiopathology and Transplantation, Center for Metrological Traceability in Laboratory Medicine (CIRME), University of Milano, Italy.

Background: A project for the standardization of HbA was launched by the IFCC back in 2004.

Materials And Methods: In this work we report on the state-of-the-art of the project on standardization of HbA. Data obtained from various EQAS studies, and from previous experimental evaluations, are presented.

Results: We have proven that biases between various commercial methods are still currently significant. We have also shown that calibration by commutable control materials may halve the inter-method variability.

Conclusions: The foundation of the reference system for HbA, together with a brief preliminary presentation of the proposed primary reference measurement procedure based on ID-MS are outlined.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295589PMC
December 2018

Determination of HbA by quantitative bottom-up proteomics and isotope dilution mass spectrometry.

Clin Chim Acta 2018 Dec 17;487:318-324. Epub 2018 Oct 17.

Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

Background: Poor comparability between laboratories is often observed in the measurement of HbA. A measurement procedure of higher metrological order is needed for value assignment to a reference material that shall be used as primary calibrator.

Method: A reference measurement procedure has been developed based on isotope dilution mass spectrometry (IDMS). The α- and δ- subunits are quantified by signature peptides released by tryptic digestion of a 25 μL-blood sample. Full length U-N-labeled HbA and HbA are used as internal standards and added to the sample at concentrations closely matching the levels of the natural forms in blood. By this, an improvement in precision could be achieved with respect to previous mass-spectrometry based methods.

Results: Recovery of HbA added to a blood sample was within 102.6-105.2%. Repeatability and within-laboratory imprecision was <2.0% for two blood samples containing HbA at a low and a high fraction. Total combined measurement uncertainty is estimated as 5.5%. Good agreement (r = 0.998) of results was obtained in a comparison of two laboratories using the described IDMS procedure. There is good correlation between commercial analytical systems and IDMS (r = 0.975-0.989). Some of the platforms provide significantly biased results, however, which potentially could be mitigated by reference to IDMS.

Conclusion: IDMS holds a promise to be suitable as a reference measurement procedure for standardization of HbA2-measurements in laboratory medicine.
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http://dx.doi.org/10.1016/j.cca.2018.10.024DOI Listing
December 2018

Sources and performance criteria of uncertainty of reference measurement procedures.

Clin Biochem 2018 Jul 29;57:29-36. Epub 2018 May 29.

Centre for Metrological Traceability in Laboratory Medicine (CIRME), Dept. of Physiopatholgy and Transplantation, Università degli Studi di Milano, Milano, Italy.

Objective: This article wants to focus on the today available Reference Measurement Procedures (RMPs) for the determination of various analytes in Laboratory Medicine and the possible tools to evaluate their performance in the laboratories who are currently using them.

Methods: A brief review on the RMPs has been performed by investigating the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. In order to evaluate their performances, we have checked the organization of three international ring trials, i.e. those regularly performed by the IFCC External Quality assessment scheme for Reference Laboratories in Laboratory Medicine (RELA), by the Center for Disease Control and Prevention (CDC) cholesterol network and by the IFCC Network for HbA.

Results: Several RMPs are available through the JCTLM database, but the best way to collect information about the RMPs and their uncertainties is to look at the reference measurement service providers (RMS). This part of the database and the background on how to listed in the database is very helpful for the assessment of expanded uncertainty (MU) and performance in general of RMPs. Worldwide, 17 RMS are listed in the database, and for most of the measurands more than one RMS is able to run the relative RMPs, with similar expanded uncertainties. As an example, for a-amylase, 4 SP offer their services with MU between 1.6 and 3.3%. In other cases (such as total cholesterol, the U may span over a broader range, i.e. from 0.02 to 3.6%). With regard to the performance evaluation, the approach is often heterogenous, and it is difficult to compare the performance of laboratories running the same RMP for the same measurand if involved in more than one EQAS.

Conclusions: The reference measurement services have been created to help laboratory professionals and manufacturers to implement the correct metrological traceability, and the JCTLM database is the only correct way to retrieve all the necessary important information to this end.
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http://dx.doi.org/10.1016/j.clinbiochem.2018.05.018DOI Listing
July 2018

Calibration by commutable control materials is able to reduce inter-method differences of current high-performance methods for HbA.

Clin Chim Acta 2018 Feb 5;477:60-65. Epub 2017 Dec 5.

Dip. di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

Background: Most of the current methods used for the determination of HbA seem not well aligned. A comparison among the best performing techniques and the commutability of some control materials currently available and under development has been evaluated.

Methods: Forty blood samples were analyzed in duplicate over two separate days by different HPLC and capillary electrophoresis systems. The commutabilities of different control materials (NIBSC WHO reagent, Bio-Rad Lyphochek, and home prepared lyophilized controls RP1-3) have been assessed by analyzing the controls in quadruplicate over two consecutive days together with the blood samples.

Results: The mean within-run imprecision of HbA measurement on blood samples (CV, %) was between 0.6% and 10.1% for HbA values <3.5%, and between 1.1 and 3.1 for HbA≥3.5%. The different methods were highly correlated (r between 0.9941 and 0.9995) although biased each other. The NIBSC WHO reagent was found not commutable in 15 over 28 comparisons, the Lyphochek 2 in 18/28, and RP3 in 4/28. Recalibration of all methods by RP1 and RP2 materials was able to reduce the overall variability from 6.8% to 3.4% at HbA≤3.0% and from 6.7% to 3.0% at HbA≥4.6%.

Conclusion: The use of adequate commutable control materials as calibrators may reduce the inter-method variability of routine methods to an extent closer to the current analytical goals of bias based on biological variability.
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http://dx.doi.org/10.1016/j.cca.2017.12.001DOI Listing
February 2018

Multicenter evaluation of an enzymatic method for glycated albumin.

Clin Chim Acta 2017 Jun 29;469:81-86. Epub 2017 Mar 29.

Dip. di Fisiopatologia Medico-Chirurgica e dei Trapianti and Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

Background: The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA test is not reliable.

Methods: We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT).

Results: The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%.

Conclusion: The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use.
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http://dx.doi.org/10.1016/j.cca.2017.03.028DOI Listing
June 2017

Glycation gap: An additional tool for glycometabolic monitoring.

Clin Chim Acta 2016 Dec 5;463:27-31. Epub 2016 Oct 5.

Dip. di Fisiopatologia medico-chirurgica e dei trapianti and Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

Background: The determination of glycated albumin (GA) has been suggested as an additional parameter having an independent added value respect to that of HbA. The determination of glycation gap (gg) has also been proposed, but few studies have addressed its potential impact in the routine evaluation of glycometabolic control.

Methods: A total of 157 subjects presenting normal whole blood cell count, no hemoglobin variants, normal creatinine levels and serum protein electrophoresis patterns were studied. In a second phase, a total of 205 subjects with no restrictions as those of the first phase study, were analyzed. HbAwas measured by capillary electrophoresis, glycated albumin by an enzymatic method and their gg were then calculated.

Results: The correlation between HbA and GA for the subjects of phase 1 was strong (r=0.8927) and significant correlation between gg and age was remarked (r=0.4486). We found 17.1% of phase 2 subjects with gg falling outside the 95% prediction intervals. Various clinical conditions seemed to affect these subjects, in our experience mostly often related to impaired renal function.

Conclusion: The glycation gap may be useful to alert clinicians about patients under unstable glycemic control or when various pre-analytical conditions my affect the reliability of the measurement of GA or HbA.
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http://dx.doi.org/10.1016/j.cca.2016.10.004DOI Listing
December 2016

Developing a reference system for the IFCC standardization of HbA.

Clin Chim Acta 2017 Apr 27;467:21-26. Epub 2016 May 27.

Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Centro per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi di Milano, Milano, Italy. Electronic address:

The importance of hemoglobin A (HbA) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA cut off values is often hampered due to poor equivalence of HbA results among methods and laboratories. Thus, the IFCC standardization program for HbA was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA standardization efforts are still in progress, including the development of a higher-order HbA reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA standardization and describe the current status of HbA testing.
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http://dx.doi.org/10.1016/j.cca.2016.05.023DOI Listing
April 2017

The role of socio-economic and clinical factors on HbA1c in children and adolescents with type 1 diabetes: an Italian multicentre survey.

Pediatr Diabetes 2017 05 16;18(3):241-248. Epub 2016 Mar 16.

Division of Paediatric Diabetes, Women's and Children's Health, AOU Ancona, Salesi Hospital, Ancona, Italy.

Objective: To identify the role of the family's socio-economic and clinical characteristics on metabolic control in children and adolescents with type 1 diabetes.

Methods: In this cross-sectional, multicentre study, 768 subjects with type 1 diabetes under 18 years of age were consecutively recruited from January 2008 to February 2009. Target condition was considered for HbA values <7.5% (<58 mmol/mol). A multiple correspondence analysis (MCA) was performed to analyze the association between the socio-economic and clinical characteristics of the participants. A logistic regression analysis was performed to identify factors associated with the subjects metabolic control. In both analyses, the family's socio-economic status was represented, measured by the Hollingshead Four-Factor Index of Social Status (SES) or by parental years of education.

Results: A total of 28.1% of subjects reached target HbA1c values. The MCA identified a strong association between at-target condition and several factors: high levels of SES or high levels of parental education, the use of the carbohydrate counting system, the use of insulin pumps, the use of the insulin delivery system over a short period of time, a normal body mass index. The logistic regression analysis showed that SES and the mother's years of education were significantly associated with the target condition [odds ratio (OR): 1.01, 95% confidence interval (CI): 1.01-1.03, p = 0.029; OR: 1.05, 95% CI: 1.01-1.10, p = 0.027, respectively).

Conclusions: Personal, clinical, and family characteristics were found to be associated with HbA target. Their identification can be crucial in addressing strategies to optimize metabolic control and improve diabetes management.
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http://dx.doi.org/10.1111/pedi.12378DOI Listing
May 2017

Performance of glycated hemoglobin (HbA(1c)) methods evaluated with EQAS studies using fresh blood samples: Still space for improvements.

Clin Chim Acta 2015 Dec 22;451(Pt B):305-9. Epub 2015 Oct 22.

Laboratorio di Standardizzazione, Servizio Medicina di Laboratorio, Ospedale San Raffaele, Milano, Italy.

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 Sebia's 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.
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http://dx.doi.org/10.1016/j.cca.2015.10.014DOI Listing
December 2015

Possible role of fructosamine 3-kinase genotyping for the management of diabetic patients.

Clin Chem Lab Med 2015 Aug;53(9):1315-20

Diabetes mellitus is a global pandemic and continues to increase in numbers and significance. Several pathogenic processes are involved in the development of such disease and these mechanisms could be influenced by genetic, epigenetic and environmental factors. Non-enzymatic glycation reactions of proteins have been strongly related to pathogenesis of chronic diabetic complications. The identification of fructosamine 3-kinase (FN3K), an enzyme involved in protein deglycation, a new form of protein repair, is of great interest. FN3K phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins, suggesting a protective role of this enzyme. Moreover, the variability in FN3K activity has been associated with some polymorphisms in the FN3K gene. Here we argue about genetic studies and evidence of FN3K involvement in diabetes, together with results of our analysis of the FN3K gene on a Caucasian cohort of diabetic patients. Present knowledge suggests that FN3K could act in concert with other molecular mechanisms and may impact on gene expression and activity of other enzymes involved in deglycation process.
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http://dx.doi.org/10.1515/cclm-2015-0207DOI Listing
August 2015

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

Clin Chim Acta 2013 Aug 13;423:1-4. Epub 2013 Apr 13.

Sezione di Biochimica Clinica, Dipartimento di Scienze della Vita e della Riproduzione, Università degli Studi di Verona, Italy.

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.
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http://dx.doi.org/10.1016/j.cca.2013.04.003DOI Listing
August 2013

Analytical goals for the determination of HbA₂.

Clin Chem Lab Med 2013 May;51(5):937-41

Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Universit a degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy.

Background: We present a study aimed to define the analytical goals for the determination of hemoglobin A₂, a minor hemoglobin present in human blood normally accounting from 2.5 % to 3.3 % of total hemoglobin, and typically increased up to 6 % – 7 % in subjects carriers of β-thalassemia.

Methods: The analytical goals have been derived using two approaches, the first one based on biologic variation, and the second one based on the opinion of experts.

Results: The data obtained by studying 17 adult non-carrier healthy subjects, from whom we took blood samples every 2 weeks for 2.0 months, indicated a small intraindividual biologic variation (CV I of 0.7 % ), with respect to a larger between-subject variation (CV G of 7.7 % ). The minimum levels for imprecision, bias and total error derived from the analysis of these data were: 0.5 % , 2.9 % and 4.5 % , respectively. The limits derived from the opinion of experts were based on a questionnaire with three clinical cases, which was circulated among two teams of international experts, and on a discussion about the clinical needs. The average total error derived from such surveys ranged between 7.0 % and 9.5 % .

Conclusions: The various methods to derive analytical performance goals gave different limits, thus indicating the need for an increased communication between clinicians and laboratory professionals on this matter.
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http://dx.doi.org/10.1515/cclm-2012-0575DOI Listing
May 2013

Reduced prevalence of the C825T polymorphism of the G-protein beta subunit gene in women with breast cancer.

Int J Biol Markers 2011 Oct-Dec;26(4):234-40

Cytogenetic Core, Women's Hospital Prof Dr José Aristodemo Pinotti - CAISM, Campinas State University (Unicamp), Campinas - Brazil.

Objective: To evaluate the prevalence of the C825T polymorphism in the GNB3 gene in women with and without breast cancer and its possible association with clinical or pathological features of breast disease.

Subjects And Methods: We included 134 women with breast cancer and a control group of 129 healthy women. The case group responded to a questionnaire on lifestyle, reproductive factors and family history. Clinical data were also evaluated. The risk for cancer was calculated and PCR was carried out for the detection of the polymorphism. Statistical analysis was performed using the package R Environment, with confidence intervals of 95% and a significance level of 5% (p and lt;0.05).

Results: The frequency of the TT genotype was significantly greater in women of the control group (30.2%) than women with breast cancer (14.9%) (p=0.02). The polymorphism was not associated with clinical features, age at diagnosis (p=0.07), age at menarche (p=0.17), and age at menopause (p=0.60). The TT genotype did not have a higher frequency in patients with high BMI (p=0.98). The risk for cancer showed no correlation with the presence of the polymorphism.

Conclusions: Our data indicate that the C825T polymorphism in the GNB3 gene has no relationship to the risk for breast cancer or the characteristics of the disease.
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http://dx.doi.org/10.5301/JBM.2011.8751DOI Listing
June 2012

The first case of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] observed in association with Hb S [β6(A3)Glu→Val, GAG>GTG] in a healthy Italian child.

Hemoglobin 2012 19;36(1):73-9. Epub 2011 Sep 19.

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Milano, Italia.

We report the first observation of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] in a Caucasian family and the first case of this variant to be found in association with Hb S [β6(A3)Glu→Val, GAG>GTG]. The proband was a healthy 4-year-old Italian boy. His chromatographic hemoglobin (Hb) pattern showed an abnormal peak having the typical retention time of Hb S (25.6% ), a second abnormal peak eluted soon after (13.6%) and a third minor peak eluted at the end of the run (6.5%). Identification of Hb variants were performed by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Two abnormal peptides at m/z 765.1 and 922 were found, corresponding to the αT-4 and βT-1 peptides characteristic for Hb G-Honolulu and Hb S, respectively. The third minor abnormal peak presumably corresponded to the hybrid molecule (α(G-Honolulu)/β(S)). The concomitant presence of Hb G-Honolulu and Hb S does not seem to produce any relevant clinical manifestation.
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http://dx.doi.org/10.3109/03630269.2011.600797DOI Listing
June 2012

Revaluation of biological variation of glycated hemoglobin (HbA(1c)) using an accurately designed protocol and an assay traceable to the IFCC reference system.

Clin Chim Acta 2011 Jul 17;412(15-16):1412-6. Epub 2011 Apr 17.

Centre for Metrological Traceability in Laboratory Medicine (CIRME), University of Milano, Milano, Italy.

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.
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http://dx.doi.org/10.1016/j.cca.2011.04.014DOI Listing
July 2011

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

Clin Chem Lab Med 2011 May 3;49(5):803-8. Epub 2011 Feb 3.

Laboratorio di Genetica Medica, Ospedale Niguarda Ca' Granda, Milano, Italy.

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.
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http://dx.doi.org/10.1515/CCLM.2011.133DOI Listing
May 2011

Towards the development of a certified reference material for hemoglobin A2.

Clin Chem Lab Med 2010 Nov 29;48(11):1611-8. Epub 2010 Oct 29.

Centre for Metrological Traceability in Laboratory Medicine (CIRME), Department of Science and Biomedical Technology, Laboratorio Interdisciplinare di Tecnologie Avanzate (LITA), University of Milan, Milan, Italy.

Background: In 2004, a working group on the standardization of hemoglobin A(2) (HbA(2)) was created within the IFCC, with the aim of developing a reference system for this analyte. One goal was to prepare a certified reference material in collaboration with the Institute for Reference Materials and Measurements (IRMM). This paper describes the properties of a first batch of this candidate study material.

Methods: Eighty millilitre of fresh whole blood, collected from a healthy blood donor, was treated by removing plasma, white blood cells and platelets. Red cells were hemolyzed to prepare 100 vials of lyophilized material (approximately 155 mg per vial). After reconstitution, the HbA(2) content was measured with a total of seven HPLC methods, three electrophoretic techniques, and two capillary electrophoresis (CE) methods. Homogeneity was tested in a subset of five vials. Stability during storage at +4°C and -20°C was tested monthly over a period of 1 year. The commutability of this material was assessed by analysing the study material together with a set of 54 fresh blood samples, with a subgroup of the above mentioned methods, only by one routine HPLC (Bio-Rad Variant II, dual kit) and by a CE method (Beckman PA800, Analis kit), respectively.

Results: The chromatographic and electrophoretic patterns obtained by all the HPLC, electrophoretic and CE techniques did not show any difference between those obtained using the first study material and those obtained with fresh blood samples. The lot was found to be homogeneous on the basis of the content of lyophilized powder per vial. The HbA(2) concentration in the lyophilized material remained stable at +4°C and -20°C, even after 1 year of storage. After reconstitution, the HbA(2) concentration did not change for more than 2 weeks in the refrigerator at +4°C. The normalized residual of the study material, measuring the degree of its commutability was 0.9, similar to that obtained on other home prepared and some commercial controls.

Conclusions: Ideally, fresh whole blood is the best reference material in the meterological traceability chain for HbA(2) analysis. However, for a number of reasons the preparation of large batches of fresh whole blood to be used as secondary reference material for HbA(2) is not practical. In our work, we have proven that lyophilization does not appear to cause any matrix effect or inhomogeneity in the study material, which also confirmed to be commutable for the Bio-Rad Variant II (dual kit) and Beckman PA800 (Analis kit) methods. We conclude that a material similarly prepared as the current study material and value assigned with the candidate reference measurement procedure still under development will be suitable to calibrate various routine methods for HbA(2). This will result in improvement of the inter-method variability for this important biochemical marker.
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http://dx.doi.org/10.1515/CCLM.2010.317DOI Listing
November 2010

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

Clin Biochem 2009 Dec 4;42(18):1797-801. Epub 2009 Jul 4.

Center for Metrological Traceability in Laboratory Medicine (CIRME) and Department of Science and Biomedical Technology, University of Milano, Milano, Italy.

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.
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http://dx.doi.org/10.1016/j.clinbiochem.2009.06.023DOI Listing
December 2009

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

Bioelectrochemistry 2008 Aug 13;73(2):137-40. Epub 2008 Apr 13.

CIRME, Dip. di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Via F.lli Cervi 93, Segrate, Italy.

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.
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http://dx.doi.org/10.1016/j.bioelechem.2008.04.010DOI Listing
August 2008

Performance characteristics and clinical utility of an enzymatic method for the measurement of glycated albumin in plasma.

Clin Biochem 2007 Dec 10;40(18):1398-405. Epub 2007 Aug 10.

Dip. di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, Italy.

Objective: The measurement of plasma glycated albumin is particularly useful in the short-middle term monitoring of glycometabolic control in diabetics. The aim of this work is to evaluate a new enzymatic method for the measurement of glycated albumin in plasma, with particular attention to some selected cases and comparison with other relevant tests (fasting plasma glucose, after glucose load, fructosamine, glycated hemoglobin).

Design And Methods: We have performed a multicenter study by which sample collection was performed in three different centers (Milano, Padova and Cagliari) and serum samples, frozen at -80 degrees C, were then delivered under dry ice to the centralized laboratory in Milano. Glycated plasma albumin was measured with reagents from Asahi Kasei Pharma (Lucica GA-L enzymatic assay; AKP, Tokyo, Japan) on a Modular P Roche system. Fructosamine was assessed by a Roche method and HbA(1c) (measured separately in the three centers on fresh EDTA blood) by DCCT-aligned HPLC systems. We have investigated 50 type 2 diabetics, 26 subjects with gestational diabetes, 35 subjects with thalassemia major, 10 subjects with cirrhosis, 23 patients with end-stage renal disease subjected to dialysis treatment and 32 healthy adult control subjects.

Results: The main analytical performance characteristics of the new GA test were the following: (a) the within-assay reproducibility was between 3.0 and 3.9% (in terms of GA% CV, measured on 2 serum pools and 2 control materials at normal and pathological glycated albumin levels); (b) the between-assays reproducibility was from 2.8 to 4.1%; (c) the linearity was tested in the interval between 13 and 36% and found acceptable (r(2)=0.9932). Concerning the clinical utility of the new test, we have evaluated the relationships between GA, HbA(1c), fructosamine and fasting and post-prandial glucose in several patients, as well as the changes in the above mentioned parameters in a sub-group of type 2 diabetic patients for 18 weeks as they progressed from severe hyperglycemia (HbA(1c) >or=10.0%) toward a better glycemic control. The correlations between glycated albumin and HbA(1c) were as follows: (a) type 2 diabetics: r(2)=0.483 (good glycemic control), r(2)=0.577 (poor control); (b) diabetic patients under dialysis: r(2)=0.480; (c) liver disease: r(2)=0.186; (d) transfused non-diabetics with thalassemia: r(2)=0.004. Glycated albumin, as well as HbA(1c) and fructosamine, was of little value in the study of women with gestational diabetes, mainly because of the very limited glucose fluctuations in this particular category of subjects. In 11 type 2 diabetic patients under poor metabolic control, GA was better correlated with fasting plasma glucose then HbA(1c) (r(2)=0.555 vs. 0.291, respectively), and decreased more rapidly than HbA(1c) during intensive insulin therapy.

Conclusions: The experience we have acquired with the new enzymatic test demonstrates its reproducibility and robustness. We confirm that plasma glycated albumin is better related to fasting plasma glucose with respect to HbA(1c). Moreover, glycated albumin is more sensitive than HbA(1c) with regard to short-term variations of glycemic control during treatment of diabetic patients. This test is also very appropriate when the interpretation of HbA(1c) is critical.
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http://dx.doi.org/10.1016/j.clinbiochem.2007.08.001DOI Listing
December 2007

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

Clin Chem Lab Med 2007 ;45(1):88-92

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi, Milano, Italy.

Background: To evaluate the extent of interlaboratory variation and accuracy in hemoglobin A(2) (HbA(2)) assays, a pilot study of external quality assessment was organized among 48 Italian laboratories routinely measuring HbA(2). As part of the study, a survey was also performed by sending a questionnaire concerning some important analytical aspects related to the determination of HbA(2).

Methods: The trial specimens consisted of three whole blood samples (A, B and C) with normal, pathological and borderline HbA(2) 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 HbA(2) levels, respectively. To assign HbA(2) target values to the samples, the median of the laboratory group was used. The accuracy of HbA(2) 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 HbA(2) should be improved.
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http://dx.doi.org/10.1515/CCLM.2007.002DOI Listing
March 2007

Reference intervals for hemoglobin A1c in pregnant women: data from an Italian multicenter study.

Clin Chem 2006 Jun 6;52(6):1138-43. Epub 2006 Apr 6.

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Segrate (Milano), Italy.

Background: The reference intervals for hemoglobin A1c (Hb A1c) in pregnant women without diabetes are not well defined, and few examples of reference intervals established by networks of different laboratories are available.

Methods: Five Italian Diabetic Care Units were involved in the study. Data were collected from 445 pregnant women without diabetes, selected on the basis of glucose challenge test results, and from 384 nonpregnant control women. The Hb A1c measurements were performed with HPLC systems aligned to the Diabetes Control and Complications Trial. Plasma glucose measurements were also performed locally. Both Hb A1c and glucose measurements were harmonized by running appropriate external quality assessment schemes. The reference intervals were calculated in terms of nonparametric 2.5th to 97.5th percentiles with 0.90 confidence intervals.

Results: The Hb A1c measurements were reproducible (CV = 2.0%) and accurate [mean (SE) difference from the target values, -0.10 (0.06)%]. Glucose measurements were also reproducible (mean CV = 3.2%) and accurate [difference from the target values, -0.01 (0.04) mmol/L]. To calculate common reference intervals, we merged the data collected in the different centers. The Hb A1c reference intervals were 4.0%-5.5% for pregnant nondiabetic women and 4.8%-6.2% for nonpregnant controls.

Conclusions: Healthy pregnant women have lower Hb A1c concentrations than nonpregnant women. The reference intervals for Hb A1c in pregnant women should therefore be lower than those currently in use.
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http://dx.doi.org/10.1373/clinchem.2005.064899DOI Listing
June 2006

Analytical evaluation of the Tosoh HLC-723 G7 automated HPLC analyzer for hemoglobin A2 and F determination.

Clin Biochem 2005 Feb;38(2):159-65

Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy.

Objectives: The analytical performance of a new automated HPLC system (Tosoh HLV-723 G7) for Hb A(2) and Hb F quantification in blood was studied.

Design And Methods: Hb A(2) and Hb F measurements were studied for imprecision, linearity, carry-over, interferences and sample concentration effect. Method comparison study was performed with the Bio-Rad Variant II HPLC system. Hb F results were also compared with those obtained by the alkaline denaturation test. The detection of some common Hb variants was also studied.

Results: Hb A(2) within-run and between-run CVs were found between 0.8-2.2% and 2.9-7.2%, respectively, while CVs for Hb F were up to 10.0% in normal and between 1.9-5.3% at more clinically relevant Hb F concentrations (>1.5%). Comparison study with Bio-Rad Variant II for Hb A(2) determination showed good correlation but highlighted calibration differences. The following results were obtained in two different laboratories: y = 1.163x - 0.52, r = 0.9918, n = 144 (Lab A); y = 1.060x - 0.40, r = 0.9920, n = 93 (Lab B). With regard to the determination of Hb F, the measurements performed by the tested method was found to correlate well with the alkaline denaturation test (y = 1.0138x - 0.36, r = 0.9842, n = 20) and with the Variant II HPLC system (y = 0.812x + 0.52, r = 0.9835, n = 110). An excellent linearity (r = 0.999) was found for both Hb A(2) and Hb F in the range 0.8-19%. Hb S, Hb C and Hb D can be presumptively identified by the assigned retention time windows.

Conclusion: The new analyzer Tosoh HLV-723 G7 was found to be a reliable method for Hb A(2) and Hb F quantification and for the presumptive identification of some common Hb variants.
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http://dx.doi.org/10.1016/j.clinbiochem.2004.10.017DOI Listing
February 2005
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