UNASSIGNED: Hemoglobin A1c has been widely used to diagnose and monitor diabetes. However, the accuracy of HbA1c analysis can be significantly affected by hemoglobin variants, leading to falsely low or elevated levels and misdiagnosis or inappropriate diabetes management.
UNASSIGNED: In this study, we present the case of a 23-year-old man with undetectable HbA1c levels during his annual checkup by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). To investigate the reason for HbA1c absence, Sanger sequencing, multiplex ligation-dependent probe amplification assay (MLPA), long-read single molecule real-time sequencing (SMRT) and MALDI-TOF mass spectrometry (MS) were performed, and the proband was identified as compound heterozygous of β-thalassemia with Hb G-Taipei (HBB:c.68A > G) and Hb Lepore-Boston-Washington (NG_000007.3:g.63632_71046del).
UNASSIGNED: The combination of these molecular technologies including MLPA, long-read SMRT sequencing and MALDI-TOF MS is beneficial for identifying rare hemoglobin variants. This case also provides essential evidence for uncovering the effect of compound heterozygosity for Hb Lepore-Boston-Washington and Hb G-Taipei on hematological phenotypes and HbA1c analysis.

Hemoglobin variants are often discovered when hemoglobin A1c (HbA1c) levels measured with a high-performance liquid chromatography (HPLC) system in fast mode are found to be low. The HA-8180V HPLC analyzer by Arkray offers two measurement modes: fast mode (FM) and variant mode (VM). Two Japanese patients with α-chain variant Hb Q-Iran detected incidentally after analyses with the HA-8180V in VM showed an abnormal peak, are presented. The first patient was a man in his 70 s, and the second patient was a man in his 50 s. Both were non-diabetic, but their results from HbA1c measurement in VM showed an abnormal peak. The VM-HbA1c, FM-HbA1c, and HbA1c measured by enzymatic assay and glycated albumin levels of the two patients were all within the reference ranges. They were diagnosed as having Hb Q-Iran (α2-75Asp → His) by globin gene analysis. It is difficult to detect α-chain hemoglobin variants based on abnormal FM-HbA1c levels, but measuring HbA1c in VM is useful for efficiently detecting hemoglobin variants.

Hemoglobin (Hb) Chile [β28(B10) Leu > Met; HBB: c.85 C > A] is a rare hemoglobin variant caused by a missense mutation in the HBB gene. Only one case of Hb Chile has been reported worldwide so far. It is an unstable hemoglobin, characterized by cyanosis associated with chronic methemoglobinemia and hemolytic anemia induced by sulfonamides or methylene blue.
A 9-year-3-month-old girl had mild anemia of unknown etiology for more than 6 years. She had a slight pallor without other symptoms or signs. The complete blood count revealed normocytic normochromic anemia with a sometimes-elevated reticulocyte count, and the bone marrow cytology showed marked erythroid hyperplasia, but the tests related to hemolysis were normal. Therefore, the whole exome sequencing was performed and showed a heterozygous mutation for HBB: c.85 C > A. With asymptomatic methemoglobinemia confirmed later, she was eventually diagnosed with Hb Chile.
This is the first report of Hb Chile in China and the second worldwide. This case shows that Hb Chile is clinically heterogeneous and difficult to diagnose and expands our understanding on the clinical and hematological traits of the disease.

UNASSIGNED: High-performance liquid chromatography (HPLC) is commonly used to measure hemoglobin A1c (HbA1c) levels and detect hemoglobin variants (Hb-Vars). HLC-723GR01 (GR01) is a new-generation automated ion-exchange HPLC system with two switchable analysis modes, namely short (30 s/test) and long modes (50 s/test). We evaluated the general performance of both analysis modes of GR01 for quantifying HbA1c and detecting Hb-Vars.
UNASSIGNED: We evaluated the instrument\'s precision based on CLSI protocol EP-05-A3. A comparison of the two analysis modes of GR01 against the standard mode of HLC-723G11 was performed on 100 whole blood samples. The GR01 long mode was compared with affinity HPLC (AF-HPLC) for detecting common Hb-Vars (HbE, HbD, HbS, and HbC, >20 samples). To examine the detection capability for minor Hb-Vars, we analyzed 26 Hb-Vars using multiple analyzers, including both analysis modes of GR01.
UNASSIGNED: Both modes of GR01 had within-laboratory coefficients of variation of ≤1.0 % from four samples with HbA1c concentrations of 32-86 mmol/mol. Good correlation was observed between GR01 and HLC-723G11. The results for HbA1c detection in the presence of the major variants revealed a strong correlation between the long mode of GR01 and AF-HPLC (r = 0.986-0.998), and the difference biases ranged 0.1-1.9 mmol/mol. In the long mode, only one variant had a difference bias exceeding 14 % [10 % (%NGSP)].
UNASSIGNED: The two analysis modes of GR01 were fast and had high accuracy and reproducibility, indicating their utility for routine clinical use in measuring HbA1c samples with Hb-Vars.

UNASSIGNED: To examine the common types of hemoglobin variants and to evaluate the influence of common variants on the results of two kinds of glycosylated hemoglobin (HbA1c) tests.
UNASSIGNED: We conducted a retrospective study, analyzing the data of a patient population undergoing two HbA1c tests, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), at West China Hospital, Sichuan University between March 2021 and February 2022. By screening the chromatograms, the hemoglobin variants were identified and their migration positions in the CE method were recorded. The effects of the variants with different migration positions on the findings of the two methods were compared. Variant samples with different migration positions were selected and Sanger sequencing was performed to determine mutations in HBA1, HBA2, and HBB genes in the variant samples.
UNASSIGNED: We examined the HbA1c of 207 786 patient samples, identifying variant peaks in the chromatograms of 372 patients. The detection rate of variants was 0.18%, with the variant identification rate of HPLC being 43.3% and that of CE, 100%. Through sequencing, 20 variants were detected. A total of 261 patient samples were tested for HbA1c with both HPLC and CE. HPLC reported all HbA1c results, while CE did not report HbA1c results for 28 samples, among which, 26 showed abnormal peaks that overlapped with HbA1c peaks, and 2 showed abnormal peaks that overlapped with HbA0 peaks. The commonly observed variant migration positions, as revealed by CE, were at the horizontal coordinates of 225±1, 200±3, 100±2, 124±1, 70±2, and 182±1. There was significant difference between HPLC method and CE method in the determination of HbA1c ( P<0.0083), and the difference between the two methods was the largest when there were variants in the 200±3 region. Linear regression showed that the correlation of HbA1c results between the two methods was different when different regional variants were present, and that the correlation between the two methods was strongest when 124±1 region was present ( r=0.998).
UNASSIGNED: There are diverse types of hemoglobin variants and most of them can affect the HbA1c findings of HPLC. Analyzing the chromatogram facilitates the identification of the variants.

UNASSIGNED: Mobility and migration flows are growing from different countries of the world to European countries, including France and in particular the Mediterranean basin. This study aimed to investigate the presence of hemoglobin (Hb) variants in outpatients/inpatients of the Montpellier Hospital (France) in whom an HbA1c assay had been performed and for which the country of birth had been informed.
UNASSIGNED: This is a retrospective study from January 2016 to December 2020 based on all high-performance liquid chromatography (HPLC) chromatograms (Tosoh Bioscience HLC-723G8) having an alarm of suspected Hb variant during HbA1c measurement. The corresponding samples were systematically sent to the hematology laboratory for confirmation and identification of Hb variant. Patient\'s medical history, clinical and demographic data were extracted from each medical chart. Statistical analyses were performed using XLSTAT® software, version 2016.06.35661.
UNASSIGNED: Three hundred sixty-three patients were confirmed with Hb variant exhibiting 17 different Hb profiles, highlighting the pivotal role of glycated hemoglobin (HbA1c) as a detection step. The prevalence of Hb variant in this southern French population was 0.71%, with the highest frequency for the beta-globin variants (n = 342/363; i.e., 94.2%), including the most common: S, C, E, and D in 200/342 (58.5%), 83/342 (24.3%), 29/342 (8.5%), and 11/342 (3.2%), respectively. Among patients with Hb variants, almost half (165/363; i.e., 45.4%) were born in the African continent with a predominance for Morocco (32/165; i.e., 19.3%) and Algeria (29/165; i.e., 17.5%).
UNASSIGNED: HbA1c assay is a useful tool to detect Hb variants. Hemoglobinopathies are a public health issue in the current French population which is a multiethnic society. Despite the monocentric nature of our study, we note a high frequency of Hb variants in the south of France, which underlines the importance of screening for Hb variants in the whole population.

Hemoglobin (Hb) variants are common factors that affect the results of glycosylated hemoglobin (A1C) tests. Hemoglobin variants react differently to different testing methods. Herein, we presented the first ever report of the effect of hemoglobin C (Hb C) on the test results of A1C in the Chinese population. High performance liquid chromatography (HPLC) and capillary electrophoresis were performed to measure A1C. Hemoglobin electrophoresis was conducted to identify the hemoglobin variants. Hb sequencing was performed to determine the mutation sites on the β chain. HPLC showed decreased A1C results, which could be corrected by electrophoresis, but the electrophoresis graph still showed abnormal peaks. The hemoglobin electrophoresis results suggested that there were hemoglobin variants, which hemoglobin sequencing results revealed to be Hb C. Uncommon variations in a specific population tend to be overlooked. To avoid clinical decision-making being affected by the results of a single test, we recommend that an explanatory reporting model be routinely adopted for A1C tests so that all reports always contain explanatory notes for the testing methodology and analysis of the graphs.

Hemoglobin A1c (HbA1c) can be unreliable (falsely elevated or lowered) in certain conditions, including hemoglobinopathies, anemia, lead poisoning, chronic alcoholism, and opioid use. Hemoglobin Wayne is a rare variant of hemoglobin (Hgb) that can also result in a false elevation of HbA1c. Hence, clinicians should be aware of these underlying causes before diagnosing and treating diabetes mellitus to avoid unexpected consequences. We are reporting a case of falsely elevated HbA1c in a female in her early 60s due to a rare variant of Hgb called hemoglobin Wayne. The patient presented with a consistently elevated HbA1c ranging from 10.3% to 10.7% for two years, which did not correlate with her fasting blood glucose levels ranging between 80 and 100. The continuous glucose monitoring (CGM) profile was also within the normal range. The hemoglobin electrophoresis technique was used to confirm the diagnosis of hemoglobin Wayne in this patient and the initial treatment of metformin was discontinued upon confirmation.

Glycated hemoglobin A1c (HbA1c) is considered the standard of care for the testing and monitoring of diabetes. Its ability to accurately reflect glycemia, however, is imperfect. Hemoglobin variants-mutant forms of hemoglobin caused by genetic variation present in 7% of the population-are known to adversely affect the ability of HbA1c measurement to reflect glycemic control. We report an illustrative case of a 64-year-old nondiabetic man with a steadily decreasing HbA1c and no symptoms of hypoglycemia or concerning family history. Preliminary investigative workup returned nothing of significance. Genetic sequencing, however, identified a rare benign hemoglobin variant: a heterozygous missense mutation in the gene encoding the hemoglobin β chain (c.155C > A, p.Pro51His). This variant has been reported only once previously, and the report predates genetic sequence data of the variant. Although this variant had no clinical implications for the patient, it was the cause of falsely low HbA1c levels on high-performance ion-exchange chromatography. This case highlights the importance of considering the effect of hemoglobin variants on the measurement of HbA1c. When available, family history should be carefully considered. Clinicians should suspect hemoglobin variants when HbA1c is too high or low, or discordant with the clinical picture.

High performance liquid chromatography (HPLC) is currently the mainstream technology for detecting hemoglobin. Glycated hemoglobin (HbA1c) is a gold indicator for diagnosing diabetes, however, the accuracy of HbA1c test is affected by thalassemia factor hemoglobin F (HbF)/hemoglobin A2 (HbA2) and variant hemoglobin during HPLC analysis. In this study, a new anti-interference hemoglobin analysis system of HPLC is proposed. In this system, the high-pressure three-gradient elution method was improved, and the particle size and sieve plate aperture in the high-pressure chromatography column and the structure of the double-plunger reciprocating series high-pressure pump were optimized. The system could diagnose both HbA1c and thalassemia factor HbF/HbA2 and variant hemoglobin, and the performance of the system was anti-interference and stable. It is expected to achieve industrialization. In this study, the HbA1c and thalassemia factor HbF/HbA2 detection performance was compared between this system and the world\'s first-line brand products such as Tosoh G8, Bio-Rad Ⅶ and D10 glycosylated hemoglobin analysis system. The results showed that the linear correlation between this system and the world-class system was good. The system is the first domestic hemoglobin analysis system by HPLC for screening of HbA1c and thalassemia factor HbF/HbA2 rapidly and accurately.
高效液相色谱法（HPLC）是目前主流的检测血红蛋白技术，糖化血红蛋白（HbA1c）是诊断糖尿病的金指标，但 HPLC 检测时 HbA1c 易受到地中海贫血因子血红蛋白 F（HbF）/血红蛋白 A2（HbA2）和血红蛋白变异体的干扰而影响检测结果的准确性。本研究基于高效液相色谱法提出一种新型抗干扰血红蛋白分析系统，本系统通过改进高压三梯度洗脱法，优化高压层析柱中微球粒径和筛板孔径，并优化双柱塞往复式串联高压泵的结构，既能检测 HbA1c 又可检测地中海贫血因子 HbF/HbA2 和血红蛋白变异体，抗干扰性好，性能稳定，有望实现产业化。本系统与日本东曹 G8、美国伯乐Ⅶ和 D10 糖化血红蛋白分析系统等世界一线品牌产品进行 HbA1c 和地中海贫血因子 HbF/HbA2 检测性能对比，结果表明，本系统与世界一流系统的线性相关性好，检测性能优异，是国内首次实现快速、精准筛查糖化血红蛋白和地中海贫血因子 HbF/HbA2 的高效液相色谱法血红蛋白分析系统。.