Incidental findings of variant hemoglobin during hemoglobin a1c testing

A variant hemoglobin fraction may be an incidental finding during HbA(1c) analysis using the G8 Tosoh HPLC analyzer, but it is unclear if the retention times and fraction patterns can reliably predict the findings of a high-performance liquid chromatography (HPLC) β-thalassemia program (Bio Rad Variant II analyzer). We chose 100 samples sent for HbA(1c) determinations (G8 Tosoh) with an incidental finding of variant hemoglobin and did a reflex test using the Bio Rad Variant II analyzer (β-thalassemia program). Two observers attempted to predict the results with that analyzer from fraction patterns and retention times of the hemoglobin variants detected with the G8. They independently identified all hemoglobin variants (HbS, Hb Setif, HbC, and HbD) by their patterns and retention times. We conclude that HPLC confirmation of certain variant hemoglobin fractions found incidentally during HbA(1c) testing on the G8 Tosoh is not necessary.     

The detection and diagnosis of hemoglobin A2' by high-performance liquid chromatography

Hemoglobin (Hb) A2' is a hematologically silent variant of HbA2 that is detected easily by high-performance liquid chromatography (HPLC), where it elutes in the S window. Our purposes were to define diagnostic criteria for the HbA2' trait using the Variant II (Bio-Rad, Hercules, CA) and to determine the prevalence of HbA2' in a metropolitan patient population. All Hb screens (N = 5,862) performed during a 26-month period were reviewed for new hemoglobinopathies. We identified 57 cases of HbA2' trait, making it the fourth most prevalent Hb variant detected in this population after HbS, HbC, and beta-thalassemia minor For HbA2' trait cases, the mean HbA2 level was 1.7% (SD, 0.17%), and the mean HbA2' level was 1.3% (SD, 0.18%). Six possible HbA2'/beta-thalassemia double heterozygotes were identified, for whom the sum of the HbA2 and HbA2' exceeded 4% of total Hb. Hb variants that might interfere with detection of HbA2' include HbS, glycosylated HbC, and HbG2. Diagnostic criteria proposed for the HbA2' trait by HPLC are HbA2 of 2% or less, S window peak of 1% to 2%, no previous diagnosis of HbS, and absence of HbG and HbC.     

Silent haemoglobin variants and determination of HbA(1c) with the HPLC Bio-Rad Variant II

AIMS: To evaluate the determination of HbA(1c) with an automated high performance liquid chromatography (HPLC) method in patients with clinically silent haemoglobin variants. METHODS: HbA(1c) values were determined with the ion exchange HPL Bio-Rad Variant II using the high resolution beta thalassaemia programme in patients with silent haemoglobin variants, namely: Hb Graz, Hb Sherwood Forest, Hb O Padova, and Hb D. RESULTS: All of these haemoglobin variants caused additional peaks in the chromatograms. No clinically useful HbA(1c) results were produced for patients with Hb Graz and Hb Sherwood Forest, the results for the patient with Hb D were too low, but the results for patients with Hb O Padova were acceptable. CONCLUSIONS: The development of this automated HPLC method modification with high resolution mode aids the identification of interference caused by the described clinically silent haemoglobin variants in HbA(1c) determination.     

Unexpected occurence of hemoglobin Athens-Georgia

The following report concerned a 47 year old Caucasian diabetic patient. Routine HPLC of HbA1c (Variant II Biorad Laboratories - hemoglobin A1c program) resulted only in the evidence of HbF (1%) and increase in HbA1c (10%). Considering the presence of HbF a standard agarose gel electrophoresis of patient's hemoglobin was performed and revealed the presence of Hb Athens-Georgia. Consequently the occurrence of HbF during determination of HbA1c by HPLC should lead to perform a standard hemoglobin electrophoresis in order to explore an hidden, unsuspected and clinically silent occurrence of rare Hb variant or additional unsuspected increase in HbA2.     

Evaluation of the new kit HbA(1c) Analyzer 2.0 Variant II Turbo (Bio-Rad)

The evaluation of glycated hemoglobin (HbA(1c)) is used in daily practice for monitoring the diabetes mellitus. HbA(1c) may be assayed by different methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new kit 2.0 available on HPLC Variant II(?) Turbo analyzer (Bio-Rad), which is characterized by an elution buffer containing sodium perchlorate. The correlation with the routine method used in our laboratory (HPLC Variant II(?) analyzer equipped with the NU kit) is good (r2?=?0.997). Intra- and inter-assay coefficients of variation are respectively lower than 0.6 and 1.6%. Linearity is excellent from 3.2% (11 mmol/mol) to 18.3% (177 mmol/mol). There is no inter-sample contamination. This method provides a result quickly (96 seconds), but does not allow separating labile HbA(1c) from carbamylated hemoglobin, contrary to the NU kit. As the control of analytical quality is a major concern for validation and clinical use of HbA(1c) results, the characteristics of this new kit make it a well-suited tool for daily laboratory practice.     

Differential diagnosis of adult hemoglobin A, F, and S conditions. A case of G gamma-beta(+)-hereditary persistence of fetal hemoglobin.

Hemoglobin (Hb) S/beta(+)-thalassemia is a hemoglobinopathy of variable but potentially severe clinical course. The condition is usually confirmed by the presence of a microcytic anemia and elevated levels of Hbs S, F, and A2 by electrophoresis. However, other less common disorders of Hb structure and synthesis may exhibit laboratory findings that mimic Hb S/beta(+)-thalassemia but have a more favorable prognosis. We present a case occurring in a man with clinical and laboratory features that were suggestive of Hb S/beta(+)-thalassemia but with normocythemia. Although nonmicrocytic variants of beta(+)-thalassemia, including concomitant nutritional deficiencies, were considered, high-pressure liquid chromatography revealed nearly all of the patient's fetal Hb to contain only G gamma chains. This pattern is most consistent with the rate but clinically benign condition of Hb S/G gamma-beta(+)-hereditary persistence of fetal Hb, a nondeletional type of hereditary persistence of fetal Hb. We discuss a diagnostic approach to adult Hb A, F, and S conditions, including thalassemias and thalassemia-like syndromes.     

Expression of hemoglobin variant migration by capillary electrophoresis relative to hemoglobin A2 improves precision

We report the precision of the mean migration position of hemoglobin (Hb)S, HbC, HbG (Philadelphia), and HbD (Los Angeles) in 193 samples of whole blood assayed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). By expressing the migration of Hb variants by CE relative to that of HbA(2) in the same sample, there was a significant improvement in the coefficient of variation for each variant studied. The potential usefulness of expressing Hb variants relative to that of HbA(2) was evaluated by comparing the separation of 2 closely migrating Hbs. When expressed by their initial migrations on CE, 25 of the 43 cases of HbG and HbD overlapped. However, when the migrations of these variants were expressed relative to the HbA(2) in the same sample, the 24 cases of HbG separated completely from the 19 cases of HbD. These findings suggest that expressing Hb variants relative to an internal standard, such as HbA(2), may be of value for establishing a library of variant Hbs evaluated by CE.     

The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method

Hemoglobin A1c (HbA1c) is an important indicator of risk for complications in patients with diabetes mellitus. Elevated fetal hemoglobin (HbF) levels have been reported to interfere with results of some HbA1c methods, but it has generally been assumed that HbA1c results from boronate-affinity methods are not affected by elevated HbF levels. None of the previous studies used the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method as the comparative HbA1c method. We, therefore, measured HbA1c in samples with normal and elevated HbF levels by several common assay methods and compared the results with those of the IFCC reference method.HbF levels of more than 20% artificially lowered HbA1c results from the Primus CLC 330/385 (Primus Diagnostics, Kansas City, MO), Siemens DCA2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), and Tosoh 2.2+ (Tosoh Bioscience, South San Francisco, CA), but not the Bio-Rad Variant II (Bio-Rad Laboratories, Hercules, CA) and Tosoh G7. Physicians and laboratory professionals need to be aware of potential interference from elevated HbF levels that could affect HbA1c results, including those from boronate-affinity methods.     

Quantitation of hemoglobin A2. An interlaboratory study.

In the 1976 hemoglobinopathy proficiency testing survey of the Center for Disease Control (CDC), whole-blood samples from hematologically normal adults and from individuals heterozygous for beta-thalassemia were shipped to survey participants. The object of this survey was to determine the state of the art for technics used to quantitate hemoglobin A2 (Hb A2) and to test the ability of laboratories to differentiate between blood samples having normal Hb A2 levels and those having elevated levels (i.e., those from individuals with beta-thalassemia trait). The results of Hb A2 quantitation obtained from 183 volunteer participant laboratories were compared with those obtained from 24 reference laboratories. Individual values varied greatly among laboratories and among methods for both normal and elevated Hb A2 samples. The results returned by many laboratories were not within 2 SD of the reference laboratory mean and also were not sufficiently accurate to differentiate between the normal blood samples and those with beta-thalassemia trait. The results suggest that methods for quantitating Hb A2 need to be standardized and a suitable method for determining laboratory performance found.     

HbA1c水平对2型糖尿病患者红细胞及胞内血红蛋白的影响

 目的: 探讨不同糖化血红蛋白(HbA1c)水平对2型糖尿病(T2DM)患者红细胞及胞内血红蛋白(Hb)的影响。方法: 选取健康人30名为对照组(H组),T2DM患者102例,按HbA1c水平分为3组:血糖控制良好组(A组)30例(HbA1c<7.0%);血糖控制较差组(B组)36例(7.0%≤HbA1c<9.0%);持续高血糖组(C组)36例(HbA1c≥9.0%)。利用新型静态显微图像分析技术和紫外-可见光谱技术,对各组红细胞及胞内Hb进行检测。结果: 与H组比较,A、B、C组红细胞的圆度因子明显增大(P<0.05)且与HbA1c水平呈正相关;B组红细胞的截面积和C组红细胞的全部形态参数均较H组明显增大(P<0.05);A、B、C组,除A、B组间红细胞长短轴参数无显著差异,其它组间参数比较差异有统计学显著性。与H组比较,A、B、C组Hb的紫外-可见光谱的谱型和谱峰未出现明显差异,但B、C组Hb的吸光度较H组明显下降(P<0.01),且C组Hb的吸光度也明显低于A组(P<0.05),随HbA1c水平的升高呈逐渐下降趋势。结论: 随HbA1c水平的升高,T2DM患者红细胞的形态发生改变,变形功能逐渐减弱,胞内Hb的结构可能发生改变。     

Interference of labile glycated hemoglobin on HbA(1c) assay by a high pressure liquid chromatography method

HbA(1c) assay by high pressure liquid chromatography remains submitted to interferences, among which that of labile HbA(1c) in 1 to 2% of samples. We have evaluated the interference of labile HbA(1c) on HbA(1c) assay using Variant II analyzer (Biorad), by in vitro formation of labile glycated haemoglobin and by evaluation of two protocols of elimination of labile HbA(1c) (wash and incubation of red blood cells in saline solution, or incubation in the wash/dilution solution of the analyzer). Levels of labile HbA(1c) higher than 4.5 % lead to underestimation of HbA(1c). The different protocols tested proved efficient and were adapted to routine conditions. The fastest method is the incubation of red blood cells in the wash/dilution solution for at least two hours, or more if labile fraction is unusually high.     

Quantitation of hemoglobin F. An interlaboratory study.

Samples of whole blood from four hematologically normal adults and from two individuals with increased fetal hemoglobin levels were shipped to laboratories participating in the 1976 and 1977 Center for Disease Control (CDC) hemoglobinopathy proficiency testing surveys. The data from these surveys were used to evaluate the interlaboratory variability of current methods used to quantitate hemoglobin F (Hb F). Results of Hb F quantitation obtained from more than 100 laboratories than voluntarily participated in the survey were compared with those obtained from 21 reference laboratories. Individual values for all samples varied greatly among laboratories and among methods. Results returned by most of the laboratories were outside two standard deviations of the reference laboratory mean and were not accurate enough to differentiate between a normal level and an increased, abnormal level.     

Evaluation of a fully automated high-performance liquid chromatography assay for hemoglobin A1c.

INTRODUCTION: Measurement of hemoglobin A1c (HbA1c) is used as an objective measure of long-term blood glucose control in diabetic patients. Recent improvements in automation combined with new recommendations for precision and accuracy have caused us to reevaluate our methods for measuring HbA1c. OBJECTIVE: We evaluated a newly automated high-performance liquid chromatography (HPLC) instrument for measurement of HbA1c (Tosoh A1c 2.2 Plus Glycohemoglobin Analyzer, Tosoh Medics, Foster City, Calif) and compared the results obtained by HPLC to those obtained with an immunoassay (Hitachi 911, Boehringer Mannheim Corporation, Indianapolis, Ind). RESULTS: The Tosoh analyzer was found to be linear in a range of 5.3% to 17% and had a throughput of 20 samples per hour. HbA1c results for 102 patient samples by the 2 techniques showed good correlation, with a slope of 0.87 and an intercept at 1.27% +/- 0.15%. Both the total and within-run coefficients of variation were consistently lower for the HPLC method compared with the immunoassay method. The HPLC method produces a chromatogram that shows the different hemoglobin fractions, allowing identification of abnormal hemoglobin variants. In heterozygous individuals, HbA1c measurements are made with no interference from the hemoglobin variant. In the case of homozygous or doubly heterozygous hemoglobin variants, the Tosoh HPLC identifies the hemoglobin variants as such and correctly does not report a HbA1c value in the presence of a markedly decreased amount of hemoglobin A. CONCLUSIONS: The Tosoh HPLC provides adequate throughput and improved precision, and the method is traceable to the Diabetes Control and Complications Trial.     

Performance of the Roche second generation hemoglobin A1c immunoassay in the presence of HB-S or HB-C traits

Blood HbA1c determination is a powerful tool for the evaluation and management of patients with diabetes mellitus. Many HbA1c analytical methods demonstrate bias in samples from patients with hemoglobinopathies. This study evaluated the analytical performance of Roche Diagnostics' 1st and 2nd generation HbA1c assays in patients with or without hemoglobinopathies whose HbA1c levels were elevated or normal, respectively. Boronate-affinity high performance liquid chromatography (HPLC) served as the reference method. Whole blood samples were collected from 80 patients with HbS or HbC whose group mean HbA1c value was elevated and also from 80 patients without hemoglobinopathy whose HbA1c values were in the well-controlled range. Each sample was assayed for HbA1c by the Primus boronate-affinity HPLC technique and by Roche's 1st and 2nd generation immunoassays using a Cobas Integra 800 analytical system. Results by the HPLC technique were compared with the results of both Roche assays by linear regression and Bland-Altman analysis. The 1st and 2nd generation assays yielded regression lines and correlation values vs HPLC assay of y = 1.43x - 1.59; R(2) = 0.83, and y = 0.94x + 0.10; R(2) = 0.92, respectively, in the 80 patients with hemoglobinopathies. The mean difference and the +/-2SD range were greater in the 1st than in the 2nd generation assay (2.68, +/-2.07 vs -0.54, +/-0.86, respectively). The 2nd generation assay also showed better performance than the 1st generation assay in samples from the 80 patients without hemoglobinopathy. In conclusion, this study validates the accuracy of Roche's 2nd generation assay, which is substantially improved over Roche's 1st generation HbA1c assay.     

一款新型糖化血红蛋白床旁检测系统线性评价

目的 对一款新型床旁检测(point-of-care testing,POCT)糖化血红蛋白(HbA1c)分析仪的线性进行评价,从而对这仪器及试剂进行初步评价.方法 根据临床和实验室标准化协会(CLSI) EP6-A文件,配制11个HbA1c浓度为等间距排列的试验样本.按随机顺序对它们进行测定,每个样本重复测定两次,对试验数据进行多项式回归分析.结果 测定结果未发现离群点.试验样本的不精密度变异系数(CVr)为2.4％.二次回归多项式非线性系数b2与0之间的差异无统计学意义(P=0.743),三次回归多项式非线性系数b2、b3与0之间的差异无统计学意义(P =0.869、0.847),最适多项式为一次多项式,最适方程为Y=0.971X +2.899(r =0.998).结论 该HbA1c检测系统在3.90％～13.45％范围内呈线性,该试剂盒申明的线性范围与本验证试验结果一致.     

High-Resolution HbA1c Separation and Hemoglobinopathy Detection With Capillary Electrophoresis

The CAPILLARYS HbA(1c) kit used on the CAPILLARYS 2 Flex Piercing system is designed for separating and quantifying the HbA(1c) fraction of hemoglobin while detecting hemoglobin variants and HbA(2). The aim of this study was to assess the performance of this new capillary electrophoresis kit for routine HbA(1c) quantification for patients with diabetes. The reliability of the system for HbA(2) measurement was also assessed. Precision, correlation, and linearity studies were performed. The effect of hemoglobin concentration and the presence of coexistent interfering substances were evaluated. The results were found to be accurate and linear in the clinically significant analytic range with excellent precision (mean interassay coefficient of variation of 0.7%). HbA(1c) measurement is independent of the presence of common hemoglobin interferences for HbA(1c) quantification and the total concentration of hemoglobin. Moreover, the technique provides a rapid and reliable separation of HbA(2). The measurement is reproducible and the results in β-thalassemia carriers were statistically different from those in noncarriers. Thus, this technique might be used for β-thalassemia detection. Given the short time of the analysis and the high throughput, this is a suitable system for the control of diabetes and the detection of hemoglobinopathies in laboratories with high workflow.     

Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin

In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-Variant beta-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state of Tennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A2 mean in Hb AS samples (n=146) is 4.09% (SD +/- 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD +/- 1.08, range 0.60 to 5.90%); and in Hb SC samples (n=27) it is 4.46% (SD +/- 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD +/- 0.25, range 2.1 to 3.0%), and the Hb A2 range in beta-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from beta-thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.     

Evaluation of the analyzer Glycomat 3000 (Biocode Hycel) for HbA1c

HbA(1c) represents a key parameter in the monitoring of glycemic balance in diabetic patients. It may be assayed by various methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new HPLC analyzer, Glycomat 3000 (Biocode Hycel). HbA(1c) values are well correlated (r(2) = 0.994) with those obtained by the HPLC analyzer used routinely in our laboratory (Variant II, Bio-Rad). However, the precision of assays can be at the limit of acceptability (CV of repeatability between 0.7 and 1.4% and CV of reproducibility between 3.3 and 4.1%) if strict conditions of calibration and quality control are not implemented. The assay is sensitive to the interference of modified fractions of hemoglobin (labile HbA(1c) and carbamylated Hb) in the clinical range, and to the presence of HbF. Besides, Glycomat 3000 is a friendly and easy to use analyzer.     

Screening for hemoglobinopathies during routine hemoglobin A1c testing using the Tosoh G7 Glycohemoglobin Analyzer

Approximately 5.1% of the US population has diabetes mellitus, and hemoglobin (Hb) A1c levels are routinely measured to monitor long-term glycemic control in these patients. Many laboratories use ion exchange chromatography for such measurements, and the presence of hemoglobin variants and hemoglobinopathies often results in abnormal peaks on the chromatogram. The goal of this study was to evaluate the potential that detection of these abnormal peaks provides as a screening tool for Hb variants and hemoglobinopathies. We examined 366 specimens with abnormal peaks observed during routine Hb A1c measurements using the G7 Glycohemoglobin Analyzer (Tosoh Bioscience, Inc.). Hb variants and hemoglobinopathies were characterized by alkaline and acid electrophoresis, solubility testing for Hb S, and clinical parameters. In 252 cases, sickle cell trait was identified with a mean retention time (RT) of 1.44 (SD +/-0.02) min. In 82 cases, Hb C trait was identified with a mean RT of 1.66 +/-0.03 min. RTs for other Hb abnormalities, including sickle cell disease, homozygous Hb C disease, C Harlem trait, alpha-chain Hb variants, Hb D trait, Hb G trait, Hb J trait, Hb Raleigh, and Hb Lepore were also determined. Our results demonstrate that routine Hb A1c testing provides a potential screening tool for the detection of common hemoglobin variants and hemoglobinopathies.The previously unreported RTs for the G7 Glycohemoglobin Analyzer are provided, which can facilitate further testing in previously undiagnosed patients and confirm the cause of abnormal peaks in patients with known hemoglobin abnormalities.     

Interference of hemoglobin Hope on beta-thalassemia diagnosis by the capillary electrophoresis Method

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.