Silent Hemoglobin Variant Research Articles

Unexpected Detection of Abnormal Hemoglobin Variants During Routine HbA1c Analysis by HPLC Technique: Challenges and Opportunities in HbA1c Testing

Background: Glycated Hemoglobin A1c (HbA1c) assay is most widely used in diabetic patients for assessing long-term control of glycemia. The presence of hemoglobin variants may be an incidental finding and can interfere with HbA1c measurements. The aim of the present study is to investigate the prevalence and impact of interference of different abnormal hemoglobin variants on HbA1c measurements during routine HbA1c testing. Methods: A total of 12,092 HbA1c samples were collected from January to August 2018. HbA1c quantification was carried out on a Variant II Bio-Rad’s HPLC analyzer. Abnormal chromatograms were further analyzed using the extended-run high-pressure liquid chromatography (HPLC) analysis in the A2/F mode. Results: The samples were examined for presence of abnormal variants. Samples producing abnormal chromatograms were further analyzed in A2/F mode to characterize hemoglobin variants. Abnormal variants were identified in 126 (1%) samples, and 74 (0.59%) sickle cell traits (SCT) were the most common variant in our findings. Moreover, 30 (0.24%) cases were eluted in the variant window in A1c mode, which on further analysis were found to be Hb E & Hb D traits. Furthermore, 3 (0.02%) cases were eluted at a RT <1 min as (unknown) and identified as Hb H. Also,19 (0.15%) samples were eluted in the P3 window at different retention times. Conclusion: Observing each chromatograph after the analysis can help us in identifying silent hemoglobin variants in routine HbA1c testing. Knowledge and awareness of common hemoglobin variants affecting measurement of HbA1c is imperative to avoid reporting of falsely low HbA1c values in diabetic population.

Hb broomhill [α1 or α2 114(GH2) pro > ala;HBA1 or HBA2:c.343C > G]: a rare Hb variant found in a diabetic chinese individual

We report a clinically silent hemoglobin (Hb) variant, Hb Broomhill [α1 or α2 114(GH2) Pro > Ala;HBA1 or HBA2:c.343C > G] in a diabetic Chinese man. The Hb fractions of the subject were analyzed using various chromatographic and electrophoretic techniques. The glycated hemoglobin (HbA1c) levels measured using cation-exchange high-performance liquid chromatography (CE-HPLC) and the boronate affinity method showed nearly identical results. Analysis of the chromatogram of the CE-HPLC revealed an abnormal shoulder peak that appeared towards the end of the elution profile. Though the capillary electrophoresis method did not interpret the results, a manual examination revealed an abnormal shoulder on the HbA0 peak. Similarly, the electropherogram of the capillary zone electrophoresis also had an abnormal shoulder on the HbA peak. A missense mutation specific to the Hb Broomhill variant was found using Sanger sequencing.

Refining Measurement of Hemoglobin A1c.

Glycation is the process by which a monosaccharide, most commonly glucose, attaches nonenzymatically to a protein. Hemoglobin A1c(Hb A1c)3 forms when hemoglobin is glycated at the N-terminal valine of the β-chain. Measurement of Hb A1c is integral to the diagnosis and management of diabetes because it reflects the average glucose (AG) over the preceding 120 days. Further, the mean Hb A1c over time determines the risk of microvascular complications of diabetes. However, a major limitation of its use is that some nonglycemic factors can influence Hb A1c concentration. A few substances (e.g., certain drugs, carbamylated hemoglobin, or labile Hb A1c) have been reported to interfere with Hb A1c measurement, but these interferences have been eliminated from most modern methods. Physiological and pathophysiological conditions reported to change Hb A1c values independently of glucose include advanced age, race, iron deficiency (with or without anemia), hemoglobin variants, and chronic kidney disease (1). In addition, many clinicians have patients with none of these factors; yet, Hb A1c values are inconsistent with glucose concentrations. Evaluation of these patients includes hemoglobin electrophoresis to identify clinically silent hemoglobin variants and measurement of erythrocyte-independent markers of chronic glycemia, such as fructosamine or glycated albumin. In several patients no explanation is identified for the inconsistency between the Hb A1c value and other measures of glycemia. Congruent with this, the A1c-Derived Average Glucose study showed that, although the relationship between Hb A1c and AG was linear, wide variation was observed between individuals (2). An Hb A1c concentration of 6.5% (the threshold for diagnosis) was associated with AG from 125 to 175 mg/dL, while Hb A1c values ranged between 5.5% and 8.0% when AG was 150 mg/dL. Interpretation of Hb A1c values is …

A Case of Hemoglobin Beckman; An example of Hemoglobin Variants Interfering with HbA1c Measurement

Aim: It has been known for a long time that the presence of clinically silent hemoglobin (Hb) variants in blood samples could influence the measurement of glycohemoglobin (HbA1c) since these abnormal hemoglobins might interfere with some methodologies. In the present study we describe the first case of Hb Beckman in Turkey. Material and Methods: The variant was detected during the HbA1c measurement by cation exchange high performance chromatography (CE-HPLC) in a patient with diabetes mellitus. Since a HbA1c result could not be obtained by this method, the test was repeated by boronate affinity HPLC. Results: Further investigation performed in order to identify the variant revealed heterozygote Hb Beckman 135 GCT-GAT (Ala --> Asp) which explained the inappropriate HbA1c result by CE-HPLC. Conclusion: This new case of Hb Beckman is thought to be a modest contribution to the subject of abnormal hemoglobins that interfere the HbA1c measurement.

Interference of hemoglobinA1c (HbA1c) detection using ion-exchange high performance liquid chromatography (HPLC) method by clinically silent hemoglobin variant in University Malaya Medical Centre (UMMC)—A case report

Background Glycated hemoglobin, measured as HbA1c is used as an index of mean glycemia in diabetic patients over the preceding 2–3 months. Various assay methods are used to measure HbA1c and many factors may interfere with its measurement according to assay method used, causing falsely high or low results. Objective To report a case of diabetic patient with clinically silent hemoglobin variant, causing undetectable HbA1c concentration using ion-exchange high performance liquid chromatography (HPLC) method. Case summary Our patient is a 65-year-old female with type 2 diabetes mellitus on diet control, hypertension and hypercholesterolemia. Her fasting blood glucose concentrations ranged from 6.2 to 7.8 mmol/L. Her HbA1c concentrations measured with immunoturbidimetry method (Cobas Integra, Roche Diagnostics) ranged from 6.11 to 7.23%, but were undetectable when measured with ion-exchange HPLC [Arkray HA8160, Diabetes Mode (also known as Menarini HA8160)]. Hemoglobin analysis identified the presence of a hemoglobin variant — Hemoglobin D Punjab. Conclusions Clinical laboratories should be aware of limitations of the HbA1c assay method used, such as potential interference with hemoglobin variant as depicted by our case. Alternative methods for monitoring glycemic control in these patients should be considered.

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

Objectives: Evaluation of HbA 1c determination with an automated ion-exchange high-performance liquid chromatography (HPLC) method in patients with clinically silent hemoglobin (Hb) variants. Design and methods: HbA 1c values were determined with the Arkray HA-8160 ion-exchange HPLC using the high-resolution, 4.2-min β-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 HbA 1c results in patients with Hb Graz and Hb Sherwood Forest, and demonstrated extra peaks with HbA 1c 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 β-thalassemia screening mode aids identification of interference due to some clinically silent Hb variants in HbA 1c determination.

Determination of glycated hemoglobin in clinically silent hemoglobin variants

Evaluation of glycated hemoglobin determination methods in patients with clinically silent hemoglobin variants. HbA1c results were determined with various methods, including a new enzymatic assay, a boronate affinity HPLC, immunoassays and ion-exchange HPLC in patients with the clinically silent hemoglobin variants Hb Graz, Hb Sherwood Forest, Hb D and Hb O Padova. The effect of hemoglobin variants on glycated hemoglobin determination was method-dependent. The enzymatic and boronate affinity HPLC method did not interfere with any of the variants evaluated. In contrast, Hb Graz interfered with all immunoassay and ion-exchange HPLC methods evaluated. The Tosoh ion-exchange HPLC method HLC-723 did not detect the late migrating Hb O Padova in the chromatogram, but this hemoglobin variant still interfered causing artificially low HbA1c results. Our study underscores the need for clinical laboratories and physicians to be aware of the limitations of their HbA1c assay methods as well as the importance of visual inspection of ion-exchange chromatograms to detect abnormalities caused by the hemoglobin variants. Samples with clinically silent Hb variants should be analyzed by a second method with a different assay principle, preferably a boronate affinity HPLC or an enzymatic assay.

Hb Yaoundé [β134(H12)Val→Ala] in Association with Hb C [β6(A3)Glu→Lys] in a Caucasian Portuguese Family

Hb Yaoundé [β134(H12)Val→Ala] is a rare, silent and asymptomatic hemoglobin (Hb) variant. It was previously reported in a Black man from Cameroon, in association with Hb Kenitra [β69(E13)Gly→Arg], and was subsequently found and described as Hb Mataro in a sub‐Saharan child. To date, Hb Yaoundé has not been described in Caucasian people and molecular studies have never been performed. Here we describe a three‐generation Caucasian Portuguese family in whom Hb Yaoundé was found in association with Hb C [β6(A3)Glu→Lys] (in the proband) and in a heterozygous state (in the proband's mother). The Hb studies of the proband's hemolysate, performed by isoelectric focusing (IEF) and low‐pressure cation exchange chromatography, only revealed an Hb variant identified as Hb C by comparison with a control. However, analysis performed by reversed‐phase high‐performance liquid chromatography (HPLC) showed two different β chain variants and a complete absence of the normal β chain. This uncommon Hb variant was identified as Hb Yaoundé by DNA sequencing analysis of the β‐globin gene (codon 134, GTG→GCG). The β‐globin gene haplotypes were determined in all family members using polymerase chain reaction (PCR)‐based methodology; Hb Yaoundé was found to be associated with the Mediterranean haplotype II. This study is the first description of Hb Yaoundé in Caucasian individuals, and its association with a Mediterranean haplotype supports the hypothesis of an independent genetic origin other than African.

HB Watford [βl(NA1)VAL→GLY]: A new, Clinically Silent Hemoglobin Variant in Linkage with a new Neutral Mutation [CAP +36 (G→A)

(2000). HB Watford [βl(NA1)VAL→GLY]: A new, Clinically Silent Hemoglobin Variant in Linkage with a new Neutral Mutation [CAP +36 (G→A)] Hemoglobin: Vol. 24, No. 4, pp. 347-353.

Hb Toranomon [β112(G14)Cys → Trp]: A New Unstable Electrophoretically Silent Hemoglobin

We describe a silent and unstable hemoglobin variant, Hb Toranomon, which was discovered by high performance liquid chromatography in an apparently healthy Japanese male during a Hb A1c assay. Isoelectrofocusing of his hemolysate and chromatographic separation of globin on a CM-cellulose column revealed no abnormality. The isopropanol precipitation test gave a positive result. Amino acid analysis of all peptides isolated from the tryptic digests of the aminoethylated and non-aminoethylated beta chain (beta A + beta X) by reversed phase high performance liquid chromatography indicated a substitution of Cys-->Trp at position 112 of the beta chain, which was confirmed by protein sequencing. cDNA sequencing showed the nucleotide sequence of the beta 112 codon to be TGG instead of TGT, confirming the amino acid substitution described above. The globin biosynthesis ratio (beta/alpha) was 1.00.

Hemoglobin variants recently detected in Austria.

Measurement of glycated hemoglobin (HbAtc) is used for routine management of diabetic patients. Glucose linkage to HbAtc reflects mean blood glucose levels during the last 3 months before examination. Various methods for HbA1c determination show abnormal values with hemoglobin variants. In some diabetic patients excessively high HbA1c values with high-performance liquid chromatography (HPLC) led to the detection of Hb Graz. In addition to Hb Graz, other silent hemoglobin variants have been found in Austria. Here we review Hb Graz, Hb Sherwood Forest, and Hb Okayama detected while using HPLC for the measurement of HbA1c in diabetic patients.

Hemoglobin Ozieri: a new α-chain variant (α71(E20)Ala → Val). Characterization using FAB- and electrospray-mass spectrometric techniques

A new silent hemoglobin variant, Hb Ozieri (α71(E20)Ala → Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the α-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the α-globin gene. This observation brings the number of variants due to a mutation in the α-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

Structural study of hemoglobin Knossos, β27 (B9) Ala→Ser: A new abnormal hemoglobin present as a silent β-thalassemia

A new electrophoretically silent hemoglobin variant is described that produces the classical phenotype of β thalassemic intermedia in association with β° thalassemia trait. This variant has the expression of a silent β thalassemia trait. The abnormal hemoglobin was detected by acid—urea—Triton—acrylamide electrophoresis and further demonstrated by isoelectric focusing. The amount of the variant in carrier is ∼30% of the total hemoglobin. No instability was found. Absence of hemoglobin A in the propositus blood facilitated structural studies. Peptides maps were normal but analysis of individual peptide spots showed an Ala→Ser substitution in the βT3. This variant has been previously called Hb Knossos (β27 (B9) Ala→Ser).

A new silent hemoglobin variant in a black family from French West Indies: Hemoglobin Le Lamentin α20 His → Gln

A new abnormal hemoglobin Hb Le Lamentin α20 (B1) His→Gln was discovered during a survey of cord blood from the French West Indies (Martinique). This variant displays an electrophoretic pattern similar to that of Hb A but can be isolated by isoelectric focusing (IEF) and Biorex 70 chromatography. Family studies showed the presence of this hemoglobin variant in the father and in two of his three children. Hematological data from the carriers were normal.