Performance Evaluation of the DxC 700 AU Chemistry Analyzer in Hemoglobin A1c Measurement

Background: Accurate measurement of glycated hemoglobin (HbA1c) is crucial for a diabetes diagnosis and subsequent patient management. The detection method and presence of variant Hb can interfere with HbA1c measurements. We evaluated the HbA1c-measuring performance of the DxC 700 AU (Beckman Coulter, Brea, CA, USA) immunoassay-based device in comparison with another immunoassay device and the reference method. Methods: A total of 120 normal and 14 variant Hb samples were analyzed using the Cobas c 513 (Roche Diagnostics, Mannheim, Germany) and DxC 700 AU analyzers. Variant Hb samples were also analyzed using the reference method, along with 20 normal samples. The accuracy, precision, linearity, and carryover were determined. Results: DxC 700 AU results strongly correlated with those of Cobas c 513 and exhibited accuracy in comparison with the reference method. The within-run, between-run, between-day, and total imprecision (%CV) values for the low- and high-concentration control materials were below 2%. The results of DxC 700 AU were linear over a wide HbA1c range (3.39%-18.30%). Although DxC 700 AU performed well in the presence of variant Hb, the HbA1c concentration was underestimated in the presence of fetal Hb. The possibility of interference from a high HbH proportion could not be ruled out. Conclusions: The overall analytical performance of DxC 700 AU was acceptable. The device is accurate, precise, and linear over a wide HbA1c concentration range. Although DxC 700 AU results highly correlated with those of Cobas c 513, caution should be exercised in cases of high HbF and HbH concentrations.

A case of α-chain variant hemoglobin (Hb Chad) with falsely high HbA1c levels measured by immunoassay.

Variant hemoglobin is often detected during the diagnosis and treatment of diabetes mellitus. We here describe a case of α2-chain variant hemoglobin (Hb Chad) that was identified as a result of differences in HbA1cs values determined by different assays. HbA1c measured by immunoassay was thus falsely high, whereas that measured by high-performance liquid chromatography (HPLC) was slightly low. Sequencing analysis revealed a heterozygous GAG (glutamic acid) → AAG (lysine) mutation at amino acid position 23 of the α2-globin gene. This residue is located at the surface of the α-chain in the crystal structure of hemoglobin. The high HbA1c value determined by immunoassay might have been the result of increased antigenicity of the variant hemoglobin, whereas the low value measured by HPLC reflected differential fractionation of the variant relative to the wild-type protein. Hb Chad has been reported in only three cases to date, and HbA1c was measured for the first time. This is the first case where falsely high HbA1c measured by immunoassay due to increased antigenicity in α-chain variant hemoglobin. This case highlights the importance of comparison with other parameters related to plasma glucose such as glycated albumin if an HbA1c abnormality is suspected. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00529-y.

3 cases of variant hemoglobin Hb A2-Niigata detected by falsely high HbA1c values.

BACKGROUND: HbA1c measurements in blood are used to monitor diabetes status. METHODS: We detected 3 cases (1 diabetic case, 1 borderline diabetic case, and 1 case with normal glucose tolerance) of variant hemoglobin Hb A2-Niigata based on falsely high HbA1c values measured by high-performance liquid chromatography (HPLC). RESULTS: In all 3 cases, HbA1c values measured by the HPLC method were higher than the reference range whereas the HbA1c values measured by immunoassay, plasma glucose and glycated albumin were within the reference range. The results of the genetic test revealed heterozygous mutation GTG (Val) → GCG (Ala) in codon 1 of the δ-globin gene in all 3 cases, based on which Hb A2-Niigata was found. Although until now Hb A2-Niigata has been reported in 3 cases, this is the first report on Hb A2-Niigata with falsely high HbA1c values. CONCLUSIONS: In this report, 3 cases of Hb A2-Niigata were found in a single institution in a short period; therefore, Hb A2-Niigata would exist frequently in a certain area. As a pathology causing falsely high HbA1c values, Hb A2-Niigata should be kept in mind.

Missing Hb Q-India Peak in a Triple-Heterozygous Patient with Hb D-Punjab/Hb Q-India/ß-Thalassemia Trait.

Interpretation of variant hemoglobins (Hbs) can pose challenges. We describe a puzzling case with multiple variant Hb peaks that was solved by family studies. A 32-year-old female with anemia and jaundice underwent cation exchange high performance liquid chromatography (HPLC), which revealed near-absence of Hb A along with variant peaks in the D- and C-windows (78.9 and 13.3%, respectively) and normal range of Hb F. As the HPLC did not fit any known pattern, family screening was performed. Her mother was heterozygous for Hb D-Punjab (HBB: c.364G>C) and Hb Q-India (HBA1: c.193G>C) with the hybrid αQ-India/ßD-Punjab eluting in the C-window on HPLC. Her sister had ß-thalassemia (ß-thal) trait, while her brother was heterozygous for Hb Q-India. In view of the family study results, the index case was interpreted as a double heterozygote for Hb D-Punjab and ß-thal with coinherited Hb Q-India. The Hb Q-India peak [retention time (RT) 4.7 min.] was absent on her HPLC as there were no normal ß-globin chains available to bind with the αQ-India chains. To the best of our knowledge, such an HPLC pattern with a missing Q-India peak, despite having inherited the αQ-India variant, has not been previously reported. This case illustrates the importance of family screening as an inexpensive and rapid method to resolve difficult and unusual HPLC patterns.

Usefulness of Plasma Glucose Adjusted #C (labile HbA1c fraction) Levels for the Screening of Variant Hemoglobin with Diabetes Mellitus.

Labile HbA1c migrates in the #C fraction with modified hemoglobin (Hb) (such as carbamylated Hb, acetaldehyde Hb, and acetylated Hb) when HbA1c is measured by Arkray's high-performance liquid chromatography (HPLC). We previously reported the usefulness of #C levels for the screening of variant Hb without diabetes mellitus. Because the #C levels are affected by plasma glucose levels, we investigated the usefulness of plasma glucose adjusted #C (PGa#C) for the screening of variant Hb complicated with diabetes mellitus. In this study, nine types of variant Hb in nine diabetic patients were included. HbA1c and the #C fraction were measured by Arkray's HPLC. Furthermore, we established a calculation formula for PGa#C by using the regression equation of #C and plasma glucose of 2,299 diabetic patients without variant Hb. If the cutoff value of PGa#C for the screening of variant Hb with diabetes mellitus was set at 1.3% or lower and 2.3% or higher, sensitivity and specificity were 89% and 99.8%, respectively. The PGa#C levels in all four slow moving variant Hb with diabetes were less than 1.3%, while the PGa#C levels of fast moving variant Hb with diabetes were abnormal values in four out of five patients [high #C level in one and low #C levels in three patients]. The screening of variant Hb with diabetes with high sensitivity and high specificity was possible by using the same cutoff values for the reference range of PGa#C as the #C values reported in non-diabetic subjects.

Two cases with Hb Andrew-Minneapolis showing high or low-normal HbA1c levels depending on the measurement method.

We experienced two cases of Hb Andrew-Minneapolis with high or low-normal HbA1c levels depending on the measurement method. Case 1 was a 25-year-old male, and case 2 was a 32-year-old pregnant woman. Both cases showed normal glucose tolerance levels and glycated albumin within the reference range. In both cases, the high-performance liquid chromatography (HPLC) method (standard mode) showed high HbA1c levels of 6.8% and 6.5%, respectively, while the HbA1c levels measured by immunoassay were low normal at 4.6% in both cases. Globin gene analysis detected heterozygous ß-chain mutations (ß144Lys → Asn) in both cases, which resulted in the diagnosis of Hb Andrew-Minneapolis. In case 1, a high-resolution HPLC chromatogram showed multiple abnormal peaks; two unknown peaks in addition to variant hemoglobin (HbX0) and glycation products of variant hemoglobin (HbX1c) were observed after in vitro glycation reaction. Although the details of unknown peaks were not identified, those might be modified hemoglobin associated with variant hemoglobin. The presence of unknown peaks could cause high HbA1c levels measured by HPLC (standard mode). Furthermore, the HbA1c level measured by immunoassay was increased to 4.9% within the reference range after adjustment for modified hemoglobin in case 1. Consequently, the high HbA1c levels measured by HPLC (standard mode) and the low-normal HbA1c level measured by immunoassay might be due to modified hemoglobin associated with variant hemoglobin.

Hb Phnom Penh Showing Falsely High or Reasonable HbA1c Values Depending on the Type of High-performance Liquid Chromatography System.

We herein report a 50-year-old Chinese woman with Hb Phnom Penh (α117Phe-Ile-α118Thr) showing high or reasonable HbA1c values depending on the type of high-performance liquid chromatography (HPLC) system. A high HbA1c value of 7.5% (HPLC assay: G9) and a reasonable HbA1c value of 5.2% (assay unknown) were observed. Therefore, the patient was refereed to our hospital; the oral glucose tolerance test showed normal glucose tolerance. The HbA1c values measured by an enzymatic assay, immunoassay, and affinity assay, as well as most HPLC assays were within the reference range, whereas those measured by the Tosoh HPLC systems were high.

IFCC-HbA1c May Be More Useful than NGSP-HbA1c in the Comparison between HbA1c Values in Variant Hemoglobin with a Mutation on α Chain and ß Chain.

HbA1c values in variant hemoglobin with a mutation on α chain and ß chain are assumed to be different because the α chain and ß chain of the globin gene have two and one gene(s), respectively. We examined whether the differentiation between HbA1c values in variant hemoglobin with a mutation on α chain (C1α) and ß chain (C1ß) is possible. Three patients with C1α and 9 patients with C1ß were included. HbA1c was measured by standard mode high-performance liquid chromatography (HPLC) (HPLC-HbA1c) and immunoassay (IA: IA-HbA1c). Glycated albumin (GA) was determined, and each ratio was compared. HbA1c is expressed in National Glycohemoglobin Standardization Program (NGSP) units (A1C) and International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units (iA1c). Both the HPLC-A1C/IA-A1C ratio and the HPLC-iA1c/IA-iA1c ratio in C1α were significantly lower than those in C1ß. Although there were no significant differences between the GA/HPLC-A1C ratios in both groups, the GA/HPLC-iA1c ratio in C1α was significantly higher than those in C1ß. If the cutoff values for the HPLC/IA-iA1c ratio and GA/HPLC-iA1c ratio were set at 65% and 5.3, respectively, both sensitivity and specificity were 100%. Differentiation between C1α and C1ß might be possible by using the HPLC-iA1c/IA-iA1c ratio or GA/HPLC-iA1c ratio.

A case of variant hemoglobin (Hb Agenogi) with type 2 diabetes mellitus showed high HbA1c levels measured by immunoassay due to enhanced antigenicity.

HbA1c is a widely utilized biomarker for the management of diabetes mellitus. The presence of variant hemoglobins might interfere with HbA1c measurement using different methods. We herein describe Hb Agenogi (ß90Glu → Lys) with type 2 diabetes mellitus whose HbA1c measured by immunoassay (IA) showed falsely high levels while HbA1c measured by high-performance liquid chromatography (HPLC) in the standard mode (SM) showed falsely low levels. To clarify the cause of the falsely high-IA-HbA1c levels, HbA1c was measured by various methods. Glycated albumin was slightly higher than the reference range. HbA1c measured by HPLC in the variant mode, affinity assay and enzymatic assay showed the range (mean ± 2SD: 6.0-6.8%) in this case. However, HbA1c measured by several HPLC-SMs showed falsely low levels (4.1-4.4%). IA-HbA1c using an antibody manufactured by Fujirebio Inc. showed falsely high levels (7.3-8.2%); whereas, IA-HbA1c using an antibody manufactured by Roche Ltd. showed the expected range (6.2-6.5%). In the case of Hb Agenogi, IA-HbA1c using an antibody manufactured by Fujirebio yielded falsely high levels. The mutation at codon 90 of the ß-globin chain might enhance antigenicity of the N-terminal peptide region and, therefore, lead to falsely high-HbA1c levels in IA-HbA1c.

Hb variants in Korea: effect on HbA1c using five routine methods.

BACKGROUND: Quantification of glycated hemoglobin (HbA1c) is a challenge in patients with hemoglobin (Hb) variants. We evaluated the impact of various Hb variants on five routine HbA1c assays by comparing with the IFCC reference measurement procedure (RMP). METHODS: Whole blood samples showing warning flags or no results on routine HPLC HbA1c assays were confirmed for Hb variants and were submitted to HbA1c quantification using Sebia Capillarys 2 Flex Piercing, Roche Tina-quant HbA1c Gen. 2, Bio-Rad Variant II Turbo 2.0, ADAMS HA-8180, Tosoh G8 standard mode, and IFCC RMP using LC-MS. RESULTS: Among 114 samples, the most common variants were Hb G-Coushatta (n=47), Queens (n=41), Ube-4 (n=11), Chad (n=4), Yamagata (n=4), G-His-Tsou (n=2), G-Taipei (n=1), Fort de France (n=1), Hoshida (n=1), and two novel variants (Hb α-globin, HBA 52 Gly>Cys and Hb ß-globin, HBB 146 His>Asn). In terms of control samples, all the result of HbA1c were "acceptable", within the criteria of ±7% compared to IFCC RMP target values. However, percentage of "unacceptable" results of samples with Hb variants were 16% for Capillarys 2, 7% for Tina-quant, 51% for Variant II Turbo 2.0, 95% for G8 standard mode, and 89% for HA-8180. The Capillarys 2 and HA-8180 assay did not provide the results in 5 and 40 samples with Hb variants, respectively. CONCLUSIONS: HbA1c results from five routine assays in patients with relatively common Hb variants in Korea showed various degrees of bias compared to those of IFCC RMP. Therefore, laboratories should be aware of the limitation of their methods with respect to interference from Hb variants found commonly in their local population and suggest an alternative HbA1c quantification method.

Identification of the Presence of Variant Hemoglobin Using a Measurement of the Labile HbA1c (#C) Fraction.

Labile HbA1c migrates in the #C fraction together with modified hemoglobin (such as carbamylated hemoglobin, acetaldehyde hemoglobin, and acetylated hemoglobin) when HbA1c is measured by Arkray's high-performance liquid chromatography (HPLC). It is assumed that most of the labile glycation products of variant hemoglobin do not migrate in #C fraction; in addition, a part of the stable glycation products of variant hemoglobin migrates in #C fraction. We hypothesized that subjects with variant hemoglobin are likely to show abnormally low or high values of #C fraction. In this study, we investigated this hypothesis. Twenty-one non-diabetic subjects with nine types of variant hemoglobin, and 103 non-diabetic subjects without variant hemoglobin were used. HbA1c and #C fraction were measured by Arkray's HPLC (HA-8180) using standard mode. The values of #C fraction in the control group were 1.75 ± 0.15% (range: 1.5-2.1%). The variant hemoglobin group reported #C fraction values of ≤1.3% in twelve subjects, ≥2.3% in five subjects, and within the reference range (1.4-2.2%) in three subjects. When the cutoff values of #C fraction were set at ≤1.3% and ≥2.3%, sensitivity and specificity were 86% and 100%, respectively. Most non-diabetic subjects with variant hemoglobin showed abnormal values of #C fraction. Measurement of #C fraction is a useful screening test for variant hemoglobin in non-diabetic subjects.

A Novel Hemoglobin Variant Associated with Congenital Erythrocytosis: Hb Seoul [ß86(F2)Ala→Thr] (HBB:c.259G>A).

We report the identification of a novel hemoglobin (Hb) variant [ß86(F2)Ala→Thr; HBB: c.259G>A], Hb Seoul, causing congenital erythrocytosis due to high oxygen affinity. The patient was a 33-year-old Korean man with isolated erythrocytosis. JAK2 somatic mutations were negative. Direct sequencing analyses revealed that the patient was heterozygous for c.259G>A, while other known causative genes (BPGM, EGLN1, EPAS, EPOR and VHL) had no mutation. ß86(F2) is a critical residue that affects the oxygen affinity. The novel variant in our patient, Hb Seoul, adds to the previously reported 4 other Hb variants from ß86(F2) substitutions that cause congenital erythrocytosis.

Diverse hematological phenotypes of ß-thalassemia carriers.

Most ß-thalassemia carriers have mild anemia, low mean corpuscular volume and mean corpuscular hemoglobin, and elevated hemoglobin α2 (HbA2 ). However, there is considerable variability resulting from coinheritance with α- and/or δ-globin gene mutations, dominant inheritance of ß-thalassemia mutations, highly unstable variant globin chains, large deletions removing part or all of the ß-globin gene cluster, loss of heterozygosity of the ß-globin gene cluster during development, or concomitant erythroid enzyme or membrane protein abnormalities. Recognition of the specific abnormality and correct diagnosis can allay anxiety and unnecessary investigation, help formulate treatment programs, and deliver appropriate genetic and family counseling.

Aldimine Formation Reaction, the First Step of the Maillard Early-phase Reaction, Might be Enhanced in Variant Hemoglobin, Hb Himeji.

Hb Himeji (ß140Ala→Asp) is known as a variant hemoglobin in which glycation is enhanced and HbA1c measured by immunoassay shows a high value. The phenomenon of enhanced glycation in Hb Himeji is based on the fact that the glycation product of variant hemoglobin (HbX1c) shows a higher value than HbA1c. In this study, we investigated whether aldimine formation reaction, the first step of the Maillard early-phase reaction, is enhanced in Hb Himeji in vitro. Three non-diabetic subjects with Hb Himeji and four non-diabetic subjects without variant hemoglobin were enrolled. In order to examine aldimine formation reaction, whole blood cells were incubated with 500 mg/dl of glucose at 37°C for 1 hour and were analyzed by high-performance liquid chromatography. Both HbA1c and HbX1c were not increased in this condition. After incubation with glucose, labile HbA1c (LA1c) fraction increased in the controls (1.1±0.3%). In subjects with Hb Himeji increases in the labile HbX1c (LX1c) fraction as well as the LA1c fraction were observed, and the degree of increase in the LX1c fraction was significantly higher than that of the LA1c fraction (1.8±0.1% vs. 0.5±0.2%, P<0.01). We have shown for the first time that aldimine (LX1c) formation reaction might be enhanced in Hb Himeji in vitro. The 140th amino acid in ß chain of hemoglobin is suggested to be involved in aldimine formation reaction.

Factors affecting A1C in non-diabetic individuals: Review and meta-analysis.

We carried out a systematic review and meta-analyses of studies that evaluated the possible effects of anemia, variant hemoglobin, and uremia on A1C levels in individuals without diabetes (DM). Medline and Embase were searched for studies that measured A1C values in groups with and without iron deficiency anemia (IDA) and/or iron deficiency (ID), variant hemoglobin and/or uremia by standardized methods. The difference between A1C levels in the groups with and without interferences was obtained by using random-effects meta-analysis and the effect size was presented as absolute difference of means (95% CI). Ten studies fulfilled the inclusion criteria, providing data from 11,176 participants without DM. There were no statistically significant differences in A1C in the presence of IDA/ID, HbS, and uremia by HPLC and uremia by immunoassay [0.79% (95% IC -0.39; 1.97), -0.13% (95% IC -0.51; 0.26), 0.15% (95% CI -0.58; 0.88) and -0.19% (95% CI -0.78; 0.40), respectively]. The effects of HbAS and uremia on A1C levels are within the expected individual variation and should not affect A1C results to diagnose DM. However, the effects of IDA/ID remain inconclusive and further studies are needed to clarify the glycation mechanisms in individuals with IDA/ID without diabetes.

Degree of discrepancy between HbA1c and glycemia in variant hemoglobin is smaller when HbA1c is measured by new-type Arkray HPLC compared with old-type HPLC.

OBJECTIVES: Although variant hemoglobin mainly demonstrates inappropriate HbA1c values measured by high-performance liquid chromatography (HPLC), these values differ depending on the HPLC model. In the 1990s, old-type HPLC models were replaced with new-type HPLC models which could separate stable HbA1c from labile HbA1c and modified hemoglobin. This study compared HbA1c values in subjects with variant hemoglobin measured using old-type Arkray HPLC (HA-8150) and new-type Arkray HPLC (HA-8160 or HA-8180). DESIGN AND METHODS: This study included non-diabetic subjects with apparently low HbA1c values who had variant hemoglobins due to a ß-chain heterozygous mutation. HbA1c was measured by old-type HPLC in 28 subjects with 12 variant hemoglobins (group 1) and new-type HPLC in six subjects with four variant hemoglobins (group 2). When HbA1c was measured by HPLC (HPLC-HbA1c), HbA1c measured by immunoassay (IA-HbA1c) and glycated albumin (GA) were also measured. RESULTS: IA-HbA1c and GA did not significantly differ between both groups. However, HPLC-HbA1c in group 2 was significantly higher than that in group 1 (group 1: 2.9 ± 0.7% vs. group 2: 3.7 ± 0.2%, P = 0.006). CONCLUSIONS: When HbA1c in subjects with variant hemoglobin is measured by new-type Arkray HPLC, the degree of discrepancy between HbA1c and glycemia is smaller compared with that measured by old-type HPLC.

Determinação de HbA1c por CLAE: interferência de variantes de hemoglobinas S e C e alta concentração de HbF / HbA1c determination by HPLC: interference of hemoglobin variants HbS, HbC and HbF high concentration

INTRODUÇÃO: O diabetes mellitus (DM) é considerado um problema importante de saúde pública; possui prevalência elevada e nos últimos anos observa-se aumento progressivo na sua incidência. OBJETIVO: verificar possíveis variações na concentração de hemoglobina (Hb) glicada (HbA1c) na presença de Hbs S e C e avaliar o impacto da redução da HbA1c na avaliação clínica e no monitoramento do paciente diabético. MATERIAL E MÉTODOS: Foram incluídos no estudo, 150 indivíduos diabéticos oriundos da cidade de Salvador, Bahia, de ambos os gêneros, com idade média de 56 anos. Foram determinadas a glicemia de jejum e a HbA1c por metodologia de oxidase-peroxidase e cromatografia líquida de alta eficiência (CLAE), respectivamente. RESULTADOS: Foram observadas variações na concentração da HbA1c em função da presença de variantes de Hb, como 7,85%, AA; 7,30%, AS e 7,15%, AC. DISCUSSÃO E CONCLUSÃO: A metodologia analítica a ser utilizada para determinação de HbA1c deve ser escolhida com base nas características gerais da população atendida e nas comorbidades associadas, pois a presença de Hbs S e C ocasiona reduções significativas de glicação. Essa redução pode levar a interpretações clínicas inadequadas relativas ao controle glicêmico dos pacientes.

INTRODUCTION: Diabetes mellitus (DM) is considered an important public health problem. It is highly prevalent and its incidence has progressively increased in recent years. OBJECTIVE: To verify possible variations of glycated hemoglobin (HbA1c) concentration in the presence of Hb S and Hb C and to evaluate the impact of HbA1c reduction on clinical evaluation and monitoring of diabetic patients. MATERIAL AND METHODS: This study comprised 150 diabetic individuals from Salvador city, Bahia, from both genders and average age of 56 years old. Fast blood glucose and HbA1c were determined by oxidase-peroxidase and high-performance liquid chromatography (HPLC) methods, respectively. RESULTS: There were variations in the concentration of HbA1c in the presence of hemoglobin variants such as AA (7.85%), AS (7.30%), and AC (7.15%). DISCUSSION AND CONCLUSION: The analytical method used to determine HbA1c needs to be chosen according to the general population characteristics and associated comorbidities, since the presence of hemoglobin S and C causes significant reductions in hemoglobin glycation, which may lead to clinical misinterpretation of patients' glycemic control.

Lack of precision in HbA(1c) values on variant II in cases of hemoglobin Q India.

This study was carried out to check the precision of HbA(1c) values on Bio-Rad Variant II in cases of a rare hemoglobin variant Q India. The study was carried out over a three month period on samples collected for HbA(1c) estimation. Seven out of eleven patients showed variable results of HbA(1c) with a very high and unacceptable intraday mean coefficient of variation (CV) of 9.93%. We conclude, that the results of HbA(1c) on Variant II can not be reported without adversely affecting HbA(1c) as a marker of long-term glycemic control in patients who have hemoglobin Q India. The HbA(1c) value of these patients needs to be assessed by a different instrument/method or the glycemic control be monitored by an alternate test like serum Fructosamine.