Assessment of the effect of hemoglobin variants on routine HbA1c measurements by electrospray ionization mass spectrometry

We applied electrospray ionization mass spectrometry (ESI-MS) to identify hemoglobin (Hb) variants, and to assess the effect of the variants on routine measurements of a glycated Hb, HbA1c. Over the past 8 years, we have diagnosed 83 cases, including 42 kinds of variant Hb, using MS as the main technology. Of these variants, 3 were new, and 9 were the first cases in Japan. Some abnormal Hbs cause diseases, and most cause erroneous values of HbA1c measured by various methods. ESI-MS was also successfully used for the accurate determination of glycated Hb. We and other groups proposed methods to examine glycated Hb by ESI-MS using enzyme-digested peptides, or intact globin without enzyme digestion. Using the peptide method, we clarified the extent of discrepancies among the HbA1c values measured by conventional methods and accurate values for samples containing various Hb variants identified by the MS method. We applied the globin method to measure the ratio of the glycated component of a variant chain and that of a normal chain obtained from the same erythrocytes. Although the glycation degree on most variant chains was similar to that on normal chains obtained from the same erythrocytes, the content of the glycated component of a particular variant beta-chain was approximately three times as much as that of the normal beta-chain. Abnormal Hbs cause erroneous values for HbA1c to various extents with commercial measurement methods, and MS may offer an unrivaled strategy to correct these errors.     

Evaluation of conditions associated with glycated hemoglobin values below the reference range

BACKGROUND: Evaluation of conditions associated with glycated hemoglobin (HbA1c) values below the reference range in HbA1c determinations. METHODS: Over a time period of 5 years, HbA1c results were determined with the ion-exchange high-performance liquid chromatography (HPLC) method HA-8140 Menarini. RESULTS: Of approximately 20 000 HbA1c results analyzed, 9 were below the reference range. The reason for HbA1c values below the reference range was found to be liver cirrhosis in 6 patients, anemia with hematological neoplasms in 2 patients, and elevated fetal hemoglobin > 1.5% in one patient. The silent hemoglobin (Hb) variant Hb Graz in 6 patients, Hb Sherwood Forest in 1 patient, homozygote HbS in one patient, and gross hypertriglyceridemia in one patient demonstrated no HbA1c result. CONCLUSIONS: In patients with liver cirrhosis, HbA1c measurements should be used with caution when evaluating long-term glucose control, and samples with suspected Hb variants should be analyzed by hemoglobin electrophoresis. Our study underscores the need for clinical laboratories and physicians to be aware of the limitations of their HbA1c assay methods as well as of the importance of visual inspection of ion-exchange chromatograms to detect HbA1c values below the reference range and abnormalities caused by the interference factors described here.     

A new hemoglobin variant found during investigations of diabetes mellitus: Hb Pavie [alpha 135 (H18) Val----Glu

Hb Pavie [alpha 135 (H18) Val----Glu], found during HbA1c measurement in a patient of Italian origin investigated for diabetes mellitus, exemplifies how the presence of an abnormal hemoglobin interferes with the measurement of glycated hemoglobin. This variant hemoglobin migrates as Hb A1c on polyacrylamide gel isoelectric focusing (IEF) and therefore hindered the estimation of glycated hemoglobin by this method. By ion-exchange high-performance liquid chromatography (IE-HPLC) Hb Pavie was eluted as a shoulder of the major component and the corresponding glycated fraction together with Hb A1b. Hb Pavie was purified in order to determine how its functional properties may modify red cell survival. The only functional abnormality observed was a slight decrease of the oxygen affinity, and therefore the total amount of glycated hemoglobin was not expected to be decreased by a shortening of the red cell survival.     

TOSOH HLC-723 G8全自动糖化血红蛋白分析仪性能评价

目的对以离子交换高效液相色谱法（HPLC）为原理的TOSOHHLC-723G8全自动糖化血红蛋白分析仪（简称HLC-723G8）检测全血糖化血红蛋白（HbA1c）的性能进行评价。方法对HLC-723G8测定HbA1c的精密度、相关性、线性、携带污染进行评价,并检测54例健康体检者的HbA1c进行参考区间验证。结果HLC-723G8测定HbA1c低、高水平批内变异系数（cv）分别为0．00％、0．47％,批间CV分别为0．76％、0．31％;HLC-723G8与HLC-723G7全自动糖化血红蛋白分析仪测定HbA1c的结果呈明显相关（F2=0．999,P〈0．001）,预期偏倚为0．06,相对偏倚值为0．83％,偏差符合率为100％;在5％～12％范围内线性良好（r2=0．9996,P〈0．001）;高值标本对低值标本的结果无明显携带污染（携带污染率为1．12％）;54名健康人群的HbA1c测定结果为4．7％-6．3％,在厂家提供的参考区间内。结论HLC-723G8测定HbA1c的性能良好,可在临床使用。     

Determination of the non-enzymatic glycation of hemoglobin by isoelectrofocusing of its globin chains

Micro cation exchange chromatography determination of HbA1c does not provide a complete picture of Hb glycation, for it does not determine all the glycated forms of hemoglobin. For the determination of total glycation, we describe here a rod IEF method, which allows the simultaneous quantitation of glycation on alpha and beta globin chains. The method exhibits good sensitivity; it is not affected by artifacts deriving from temperature, hypertriglyceridemia, Hb variants or labile HbA1 (aldiminic Hb). The results obtained indicate that in a normal population approximately 18% of the beta chain and 8% of the alpha chain are glycated. These mean percentages increase in the diabetic to 28% and 12%, respectively. The beta chain is glycated on both valine and lysine residues, while the alpha chain is glycated only on the latter. HbA1 values from micro cation exchange chromatography are significantly related to both alpha and beta glycation. Thus, valinic or lysinic glycation have roughly the same clinical significance.     

Silent hemoglobin variants and determination of HbA(1c) with the high-resolution program of the HPLC HA-8160 hemoglobin analyzer

OBJECTIVES: Evaluation of HbA1c determination with an automated ion-exchange high-performance liquid chromatography (HPLC) method in patients with clinically silent hemoglobin (Hb) variants. DESIGN AND METHODS: HbA1c values were determined with the Arkray HA-8160 ion-exchange HPLC using the high-resolution, 4.2-min beta-thalassemia screening mode in patients with silent hemoglobin (Hb) variants, namely, Hb Graz, Hb Sherwood Forest, Hb O Padova, and HbD. RESULTS: All of these hemoglobin variants caused additional peaks in the chromatograms, without HbA1c results in patients with Hb Graz and Hb Sherwood Forest, and demonstrated extra peaks with HbA1c results that were clinically too low for patients with Hb O Padova and in the patient with HbD. CONCLUSIONS: The development of this automated HPLC method modification with high-resolution beta-thalassemia screening mode aids identification of interference due to some clinically silent Hb variants in HbA(1c) determination.     

Multicenter evaluation of Tosoh glycohemoglobin analyzer.

BACKGROUND: We describe an Anglo-French evaluation of a new analyzer. METHODS: The Tosoh HLC-723 GHb V, A1c2.2 glycohemoglobin analyzer is an HPLC instrument with primary blood tube sampling, bar-code reading, cap piercing, and the ability to chromatographically separate labile hemoglobin A1c (HbA1c). We evaluated two analytical protocols, 2.2 and 3.0 min, and compared results for blood samples collected from diabetic and nondiabetic subjects with those obtained with Bio-Rad Diamat and Variant analyzers. RESULTS: Within- and between batch-imprecision (CVs) was <2% with linearity to at least 15.9% HbA1c. Although some hemoglobinopathies were detected in the 2. 2-min chromatography, clearer evidence of abnormality was visible in the 3.0-min version. Comparison with established methods showed good correlation (r = 0.993; n = 316 with Diamat; and r = 0.995; n = 133 with Variant) but highlighted calibration differences. CONCLUSIONS: The problems of manual blood sample preparation, labile HbA1c, and carbamylated hemoglobin interference associated with the older instruments have been eliminated in the new Tosoh analyzer. The 3. 0-min protocol is preferred for routine use.     

HLC-723 G7糖化血红蛋白分析仪性能的评价及其在糖尿病筛查的应用

目的评价HLC-723 G7糖化血红蛋白分析仪的临床应用性能,并建立深圳地区健康人群糖化血红蛋白（HbA1c）的参考区间及用于糖尿病筛查的＂cut-off＂值。方法对HLC-723 G7测定HbA1c的精密度、准确度、线性、携带污染进行评价;并检测482例健康体检者和150例糖尿病患者HbA1c。结果HLC-723 G7测定HbA1c高、低两水平批内精密度的CV分别为0.69%、0.94%,总精密度的CV分别为1.25%、1.79%;HLC-723 G7与VARIANTⅡ测定HbA1c结果呈明显相关（r=0.996,Sy.x=0.28,P〈0.001）,在6.0%、7.0%、9.0%的相对偏倚分别为-0.40%-、0.14%、0.20%;HLC-723 G7测定HbA1c在3.1%-15.9%范围内线性良好（r=0.999,P〈0.001）,高值标本对低值标本的测定结果无明显携带污染;深圳地区健康人群HbA1c总体参考区间为4.5%-6.1%;HbA1c用于糖尿病筛查受试者工作（ROC）曲线下面积为0.934,＂cut-off＂值5.9%处灵敏度为82.0%,特异性为91.3%。结论HLC-723 G7糖化血红蛋白分析仪测定HbA1c性能良好,可用于糖尿病的早期筛查。     

Analytical evaluation of the Tosoh HLC-723 G8 automated HPLC analyzer for hemoglobin analysis in beta-thalassemia mode

ObjectivesThe analytical performances of a new kit conceived for Hb variants separation and measurement procedures on an HPLC instrument (Tosoh HLC-723 G8) were studied.ResultsBetween-run and within-run precision tests were satisfactory for both HbA2 and HbF measurements. HbA2 and HbF values measured using the TOSOH HLC-723 G8 were correlated to those obtained using the Bio-Rad Variant II HPLC system (r = 0.974 and 0.997 respectively) and to those given by the Sebia Capillary's system (r = 0.980 and 0.996 respectively). Linearity was observed for HbA2 from 2.24% to 6.56% and for HbF from 0.5 to 6%.ConclusionThe new analyzer Tosoh HLC-723 G8 was found to have a wide analytical range for both HbA2 and HbF. The G8 Mode beta-thal is suitable for a first-level laboratory screening for Hb analysis but also for a second-level test for the characterization of Hb variants after a first-line screening.     

Capillary electrophoresis of hemoglobin.

Capillary electrophoresis (CE) has been used in a variety of in-house capillary isoelectric focusing (CIEF) and capillary zone electrophoresis (CZE) assays for the detection of hemoglobin (Hb) variants and the quantitation of HbA2 and HbF. A commercial kit has also been produced for the analysis of hemoglobin variants and thalassemia screening. Though CE methods have been shown to be able to detect many variants, final identification of the variant needs specialized testing such as DNA technology. Over the past 2 years, many instruments that had been used for these hemoglobin variant screening and thalassemia assays have been withdrawn from sale. Although CE HbA1c analysis is available, it cannot compete in turnaround time or cost with automated HPLC commercial instruments that give accurate HbA1c results in 3 or 4 minutes. Hence we do not anticipate a bright future for the analysis of hemoglobin by CE.     

三种相同原理的糖化血红蛋白分析仪检测结果的初步比对

目的比较3种常用的基于离子交换高效液相色谱原理的糖化血红蛋白(HbA1c)分析仪[TOSOH HLC-723 G7全自动HbA1c分析仪(简称HLC-723 G7)、Bio-Rad D-10全自动HbA1c分析仪(简称D-10)和ArkrayADAMSTMA1c HA-8160全自动HbA1c分析仪(简称HA-8160)]测定商品质控品和临床患者样本的结果,以评价不同仪器测定结果间的偏差。方法于13 d内分别使用3种仪器测定高、低浓度HbA1c质控品,合格后测定58份临床患者新鲜样本。使用Levene’s检验对3种仪器测定质控品和临床样本的结果进行方差齐性检验;认定方差相等后,对其进行单因素方差分析,观察3种仪器结果间的差异;根据临床样本结果,分别作散点图及偏差图用以观察3种仪器之间测定结果的相关性及偏差;分别计算每2个仪器间的线性回归方程,并对患者样本HbA1c结果进行预期偏差估计。结果 3种仪器测定低值质控品的结果差异有统计学意义(P<0.05),测定高值质控品的结果差异无统计学意义(P>0.05)。3种仪器临床样本的测定结果之间相关性较好(P>0.05)。在4.9%～11.4%HbA1c范围内,HA-8160、D-10的线性回归方程为Y=0.963X+0.179,r=0.988,r2=0.976;测定HbA1c的相对偏差为-0.37%～0.72%。HLC-723 G7、D-10的线性回归方程为Y=0.968X+0.523,r=0.991,r2=0.982;测定HbA1c的相对偏差为-0.1%～0.8%。HLC-723 G7、HA-8160的线性回归方程为Y=0.987X+0.480,r=0.984,r2=0.969;测定HbA1c的相对偏差为-0.13%～1.12%。结论在常见的生理及病理范围内,HLC-723G7、D-10和HA-8160 3种仪器的测定结果之间没有明显偏差。     

Long-term evaluation of electrospray ionization mass spectrometric analysis of glycated hemoglobin

BACKGROUND: Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb alpha and beta chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment. METHODS: We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini). RESULTS: Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6-5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the alpha chain relative to the beta chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of alpha- and beta-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the ss-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5-10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values. CONCLUSIONS: ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the beta chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.     

Effect of HbS in the determination of HbA(2) with the TOSOH HLC-723G7 analyzer and the HELENA Beta-Thal Quik column kit

OBJECTIVES: The analytical performance of the TOSOH HLC-723G7 hemoglobin HPLC analyzer and the effect of the presence of HbS in the determination of HbA(2) using HPLC and manual column methods. DESIGN AND METHODS: The performance characteristics of the TOSOH HLC-723G7 analyzer in the determination of HbA(2) were compared to those of the HELENA Beta-Thal Quik column. The effect of HbS presence in the samples was quantified using the HELENA SAS-MX alkaline gel electrophoresis kit as the reference method. RESULTS: Within-run and between-run CVs for HbA(2) were better for the TOSOH HPLC analyzer than for the HELENA manual column method. The presence of HbS in the samples produces a strong positive bias in the %HbA(2) values when using both the HPLC and manual column methods, compared to the alkaline electrophoresis gel. CONCLUSION: Both the TOSOH HPLC and the manual column are reliable methods for %HbA(2) determination when no HbS is detectable in the samples. When HbS is present, the gel electrophoresis method gives more accurate results.     

Development of a high-resolution melting method for the detection of hemoglobin alpha variants

ObjectivesThe present study was aimed at identifying hemoglobin (Hb) alpha variants.Design and methodsTo identify Hb variants, a high-resolution melting (HRM) method was performed.ResultsThe diagnostic strategy was found to be successful in identifying Hb alpha variants including HBA1:c.27G>T, (Hb Hekinan) HBA1:c.46G>C (Hb Ottawa), HBA2:c.31_33AG (Hb α2-globin gene codon del AG), HBA1:c.223G>C (Hb G-Taichung), HBA1:p.Phe118_Thr119insIle (Hb Phnom Penh), HBA2:c.369C>G (Hb Westmead), HBA2:c.364G>A (or HBA1) (Hb Owari), HBA2:c.377T>C (Hb Quong Sze), and HBA2:c.427T>C (Hb Constant Spring). Each Hb variant could be readily and easily identified through the difference in plotted curves. In addition, the Hb variants could be distinguished to be located at either HBA1 or HBA2 gene.ConclusionsThe HRM analysis is found to be a good tool for identifying Hb variants in alpha globin genes.     

Evaluation of HbA1c determination methods in patients with hemoglobinopathies

OBJECTIVE: To evaluate commercially available determination methods for HbA1c in patients with hemoglobin variants. RESEARCH DESIGN AND METHODS: HbA1c values were determined with various commercially available methods, including ion-exchange high-performance liquid chromatography (HPLC), boronate affinity assay, and immunoagglutination in patients with the hemoglobin mutations Hb Graz, Hb Sherwood Forest, Hb O Padova, Hb D, and Hb S. RESULTS: The effect of hemoglobinopathies on glycohemoglobin measurements was highly method dependent. The HPLC methods for HbA1c determination lacked the resolution necessary to differentiate hemoglobin variants. They demonstrated additional peaks in the chromatograms and HbA1c results either too low or too high compared with the nondiabetic reference range. With all immunoassays, Hb Graz demonstrated falsely low values. The other hemoglobinopathies in our study caused falsely low and/or high HbA1c results in immunoagglutination methods. The boronate affinity method showed values in an acceptable range for all hemoglobin variants. CONCLUSIONS: Because of the local occurrence of Hb variants and the ethnic origin of a given population, every individual laboratory must establish and validate its own assay method. In managing diabetic patients, knowledge of hemoglobinopathies influencing HbA1c determination methods is essential because hemoglobin variants could cause mismanagement of diabetes resulting from false HbA1c determinations.     

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.     

Hb Maruchi [α165 (E14) Ala>Pro; HBA1: c.196G>C]: A new thalassemia hemoglobinopathy related to the alpha1 globin gene

ObjectiveTo describe a new mutation causing alpha thalassemia and its mechanism of action.Design and methodsThe propositus was a 37-year-old man who presented maintained microcytosis without iron deficiency. Molecular characterization was undertaken using automatic sequencing after testing negative for the most frequent α-globin mutations by multiplex PCR followed by reverse-hybridization.ResultsThe mutation is a single base substitution at codon 65 of the α1 globin gene [α65(E14) Ala>Pro; HBA1: c.196G>C] and leads to the substitution of a proline residue in the E helix. The resulting hemoglobin variant has been named Hb Maruchi. This new variant cannot be separated from Hb A by electrophoretic and chromatographic techniques.ConclusionsThe substitution α65(E14) Ala>Pro; HBA1: c.196G>C causes a α-thalassemia silent associated with a very mild phenotype. The diagnosis of this type of mutation is important because it may cause alpha thalassemia if inherited with other clinically relevant HBA1/HBA2 variants.     

Quantification of hemoglobin A2 and identification of hemoglobin variants using a fully automated hemoglobin analyzer

The DIAMAT Analyser System is a fully automated high performance liquid chromatographic (HPLC) instrument originally designed for the quantification of glycated hemoglobin (HbA1c). Buffers were developed for the separation and quantification of hemoglobin A2 on the DIAMAT. Also studied were the retention patterns of various hemoglobin variants on the DIAMAT using the buffers developed for the hemoglobin A2 quantification.     

Tentative identification of hemoglobin variants in the Bio-Rad VARIANT II Hb A1C method

OBJECTIVE: To evaluate if peak characteristics, the retention time, and percentage of total hemoglobin of an unknown peak on the Bio-Rad VARIANT II Hb A1c method may be used to establish a tentative identification of hemoglobin variants. METHOD: The peak characteristics, retention time, and percentage of total hemoglobin obtained on abnormal peaks found on the Bio-Rad VARIANT II Hb A1c method were tabulated and evaluated against the identification of the hemoglobin variant established by the Bio-Rad beta thalassemia HPLC method and hemoglobin electrophoresis at both acid and alkaline pH. RESULTS: Some hemoglobin variants show specific peak characteristics, retention times, and percentage of total hemoglobin on the Bio-Rad VARIANT II Hb A1c method that allows for tentative identification of the hemoglobin variant. The retention times and percentage of hemoglobin variant for the common hemoglobin variants E, D, S, and C obtained on the Bio-Rad VARIANT II Hb A1c method are tabulated below. CONCLUSION: Peak characteristics, retention times, and percentage of hemoglobin variant may be used to establish the presence of a hemoglobin variant and to provide a tentative identification of some hemoglobin variants on the Bio-Rad VARIANT II Hb A1c method.