False HbA1cValue due to a Rare Variant of Hemoglobin J-Cubujuqui.

BACKGROUND: Hemoglobin (Hb) J-Cubujuqui is a rare Hb variant, and reports about it are very limited. There are no descriptions that it affects the results of glycated Hb. METHODS: In this study, we describe a rare variant discovered during newborn screening. Both high-performance liquid chromatography (HPLC) and capillary electrophoresis for hemoglobin analysis displayed abnormal peaks. The Hb variant was confirmed by Sanger sequencing. RESULTS: The pedigree study shows the variant was inherited from the newborn's father. His fasting blood glucose (FBG) level was 5.5 mmol/L. HbA1c measured by HPLC was falsely low in her father (2.41%), whereas that measured by immunoassay was normal (5.11%). Sanger sequencing revealed a heterozygous mutation (CGT˃AGT) at amino acid position 141 of the α1 gene, corresponding to Hb J-Cubujuqui [α1 141(HC3) Arg→Ser (CGT˃AGT); HBA1:c.424C˃A (or HBA2)]. CONCLUSIONS: This is the first report that Hb J-Cubujuqui interferes with the measurement of HbA1cand prompts clinicians to pay attention to the accuracy of glycated Hb results.

Eleven Cases of Hb J-Paris-I [HBA2: c.38C>A (or HBA1)]: A Stable α Chain Variant Elutes in the P3 Window on High-Performance Liquid Chromatography.

Hb J-Paris-I [HBA2: c.38C>A (or HBA1)] is a stable fast-moving hemoglobin (Hb) that elutes in the P3 window on high performance liquid chromatography (HPLC). The mutation can happen on either the α1- or α2-globin gene. Codon 12 changes from GCC to GAC to replace the alanine amino acid with aspartic acid. This change is external with no clinical significance. The elution in the P3 wave on HPLC can interfere with the glycated Hb assay by HPLC. In this study, data of 11 cases of Hb J-Paris-I were thoroughly presented. The majority of the cases were of Indian ethnicity. The mean value of Hb J-Paris-I on HPLC was 26.7 ± 2.0%. The retention time (RT) was 1.75 ± 0.03 min. The isoelectric focusing (IEF) mean value was -5.6 (range -6.1 to -4.9). Hb A2 was consistently reduced to 1.8 ± 0.3%. A fraction of 0.8% corresponding to the Hb A2-J-Paris-I (α2J-Paris-Iδ2) is likely to be concealed within the A0 peak of Hb A on HPLC. Interestingly, two cases were associated with two different polymorphisms [HBA2: c.-24C>G or Cap +14 (C>G) and HBA2: c.*136A>G polymorphism] without apparent effect on the variant expression.

Unexpected HbA1c results in the presence of three rare hemoglobin variants.

Hemoglobin (Hb) variants, characterized by structural abnormalities in the globin chains, are among the most common inherited disorders. It has been shown that Hb variant remains an important cause of erroneous HbA1c results. Thus, it is important to be aware of the extent of the interference of each Hb variant encountered to avoid reporting unreliable results. However, the effects of many types of Hb variants on the measurement of HbA1c remain unclear. Here, we describe three rare Hb variants, Hb J-Tashikuergan [HBA2: c.59 C > A], Hb Pyrgos [HBB: c.251G > A], and Hb Hope [HBB: c.410 G > A], which lead to extremely high values (>25%) determined by Variant II Turbo 2.0. We further investigated their effects on HbA1c measurement by an HPLC system (Bio-Rad D100), a CE system (Sebia Capillarys 3 TERA), a boronate affinity chromatography system (Premier Hb9210), and an immunoassay method (Roche Diagnostics), and found that these Hb variants severely interfered with HbA1c measurement by Variant II Turbo 2.0 and Bio-Rad D100. This study demonstrates that patients with abnormally high HbA1c levels should be highly suspected of carrying Hb variants. When the HbA1c results are considered unreliable, other indicators such as glycated albumin may be a possible alternative to HbA1c in diabetic patients.

Fifteen Cases of Hb J-Meerut: The Rare Association with Hb E and/or HBA1: c.-24C>G (or HBA2) Variants.

Hb J-Meerut [HBA2: c.362C>A (or HBA1)] is a rare, stable, nonpathogenic α-globin gene variant that peaks in the area between the P3 and A0 windows on high performance liquid chromatography (HPLC). Few cases from different ethnic origins have been published but the majority were Asian Indians. Coinheritance with other hemoglobin (Hb) variants are rarer and can change the Hb J-Meerut phenotype making a diagnostic dilemma. In this study, we have reported 15 cases of Hb J-Meerut, discovered during a wide spectrum study of α-globin chain variants in the UK. The diagnosis was confirmed by forward and reverse DNA sequencing of the α1- and α2-globin genes. The average of the Hb J-Meerut expression was 20.9% of total Hb and characterized by a retention time (RT) of 1.9 min. (on average) on HPLC. The median of isoelectric focusing (IEF) was 5.6 mm above Hb A. Among the 15 cases studied, one case coinherited the Hb E (HBB: c.79G>A) mutation in heterozygosity and another case was associated with the Cap +14 (C>G) [HBA1: c.-24C>G (or HBA2)] variant. We noticed that the coinheritance of the Hb E mutation reduced the Hb J-Meerut expression with the formation of a hybrid peak missed on the HPLC chromatograph. We also noticed an increased expression of Hb J-Meerut in the case showing the coinheritance of the HBA2: c.-24C>G (or HBA1) variant.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient.

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

Evaluation of the Interference of Hemoglobin Variant J-Bangkok on Glycated Hemoglobin (HbA1c) Measurement by Five Different Methods.

Objective The interference of the hemoglobin variant (Hb J-Bangkok) was evaluated on 4 different glycated hemoglobin assays and compared with a reference immuno assay. Methods An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of Hb J-Bangkok caused a statistically significant difference in HbA1c results compared with a reference immuno assay. Statistical analysis was performed on the difference of the estimated average glucose calculated from HbA1c values and fasting plasma glucose in the Hb J-Bangkok variant group using the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok had a significant interference on HbA1c results using an HbA1c±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results Turbidimetric inhibition immunoassay method, and enzymatic methods were not affected by Hb J-Bangkok. However, Hb J-Bangkok showed statistically significant interference to the two ion-exchange high-performance liquid chromatography methods. Conclusion When performing HbA1c tests, clinical laboratory personnel should identify the Hb variant and select the appropriate methods or use alternative indicators.

Haemoglobin J-Baltimore can be detected by HbA1c electropherogram but with underestimated HbA1c value.

Glycated haemoglobin (HbA(1c)) is considered the gold standard for assessing diabetes compensation and treatment. In addition, fortuitous detection of haemoglobin variants during HbA1c measurement is not rare. Recently, two publications reported different conclusions on accuracy of HbA(1c) value using capillary electrophoresis method in presence of haemoglobin J-Baltimore (HbJ).Here we describe the fortuitous detection of unknown HbJ using capillary electrophoresis for measurement of HbA(1c). A patient followed for gestational diabetes in our laboratory presented unknown haemoglobin on Capillarys 2 Flex Piercing analyser which was identified as HbJ. HbJ is not associated with haematological abnormalities. High Performance Liquid Chromatography methods are known to possibly underestimate HbA(1c) value in the presence of this variant. This variant and its glycated form are clearly distinguished on electropherogram but HbJ was responsible for underestimating the true area of HbA(1c). Capillary electrophoresis is a good method for detecting HbJ but does not seem suitable for evaluation of HbA(1C) value in patients in presence of HbJ variant.

[Effects of hemoglobin J-Bangkok traits on measurements of glycated hemoglobin by five methods].

OBJECTIVE: To evaluate the interference of hemoglobin variants J-Bangkok on glycated hemoglobin (HbA(1)c) detected by five measurement systems. METHODS: Seventy cases of blood samples were collected at Zhongshan Hospital of Sun Yat-sen University from July 2012 to January 2014, the blood samples were divided into the normal control group (40 cases) and Hb J-Bangkok variant group (30 cases), and the normal control group was divided into healthy control group (20 cases) and diabetic group (20 cases). HbA(1)c measurement systems were Primus Ultra2, Variant Ⅱ, Variant Ⅱ Turbo, Modular P and Leadman. Based on the standard of the American National Glycosylated Hemoglobin Standardization Program (NGSP), Primus Ultra2 was used as comparative system, and the other 4 systems were test systems. Comparative analysis and bias evaluation were conducted on the results from five detection systems in different groups, statistical analysis were used for evaluating the differences. The estimated average glucose (eAG) was calculated by HbA(1)c values and fasting plasma glucose (FPG) of Hb J-Bangkok variant group with the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok produced significant clinical effect on HbA(1)c results. HbA(1)c ± 10% and relative bias at 6% and 9% HbA1c were evaluation limits. RESULTS: The differences of the 95% confidence interval (95%CI) between the test systems and the comparative system in control group were within ±0.7% HbA(1)c, bias were less than 6%, there were no statistically significant difference (P>0.05). In Hb J-Bangkok group, the eAG calculated from HbA(1)c measured by using Primus Ultra2, Modular P and Leadman were (8.14±2.99), (8.10±3.06) and (8.23±3.00)mmol/L, which had no statistically significant difference compared with FPG ((8.21±3.12)mmol/L, t=0.996, 1.091, 1.479, all P>0.05), and the differences of 95%CI between the results measured by Modular P and the comparative system were all within ±0.7% HbA(1)c, bias were -4.3%-0.4% and -5.2%-4.9%, there were no statistically significant difference (P>0.05). At 6% and 9% HbA(1)c concentrations, the mean differences of the results from the three detection systems were less than the clinically acceptable range. These results showed that the systems of Primus Ultra2, Modular P and Leadman were not affected by Hb J-Bangkok. However, the eAG values calculated from HbA(1)c of Variant Ⅱ and Variant Ⅱ Turbo were (5.58±2.12) and(5.00±2.13)mmol/L, which showed statistically significant lower results compared with FPG level (t=12.29, 13.23 , all P<0.001). Compared with Primus Ultra2, the differences of 95%CI were outside of ± 0.7% HbA1c, bias were -31.9%--12.0% and -42.0%- -17.6% , greater than 6%, showed a negative bias.At 6% and 9% HbA(1)c concentrations, the mean differences of the results were all greater than the clinical acceptable range. These results indicated that Hb J-Bangkok had significantly clinical interference on Variant Ⅱ and Variant Ⅱ Turbo systems. CONCLUSION: Hb J-Bangkok has different interference on different HbA(1)c measurement systems, when performs the HbA(1)c test, clinical laboratory should pay attention to identify Hb variants, and select the appropriate methods to measure the HbA(1)c values in order to prevent the occurrence of interference by Hb variants.

First Report of a Chinese Family Carrying a Double Heterozygosity for Hb Q-Thailand and Hb J-Bangkok.

The double heterozygosity for α and ß chain variants leads to the formation of abnormal heterodimer hybrids, which could render laboratory diagnostics in a routine setting difficult. The following is the first report of a double heterozygosity for Hb Q-Thailand [α74(EF3)Asp→His; HBA1: c.223G>C] with α+-thalassemia (α+-thal) and Hb J-Bangkok [ß56(D7)Gly→Asp; HBB: c.170G>A] found in a Chinese family. Both subjects were healthy with normal or borderline hematological parameters. Hemoglobin (Hb) analyses showed a novel variant, Hb Q-Thailand and Hb J-Bangkok. Family studies helped in the initial recognition and in making presumptive diagnoses, but definitive diagnoses of these cases with complex α and ß chain variants could only be obtained after DNA analysis.

Hemoglobin Variants Acquired Post-Exchange Transfusion in Pediatric Sickle Cell Disease (SCD) Patients.

BACKGROUND: Many SCD patients receive chronic transfusions for prevention or treatment of disease related complications. Complications of the chronic transfusion noted in these patients include allergic reactions, transfusion transmitted infections, iron overload, and alloantibody formation. Even though hemoglobin (Hb) variants are prevalent in the general population, reports of transfusion-acquired Hb variants are rare. We performed a retrospective analysis on all SCD patients who underwent red cell exchange (RBCEx) transfusions at our institution during 2011-2013 to identify the presence of Hb variants acquired as a result of RBCEx. RESULTS: We found 66 occurrences of acquired Hb variants in 30 SCD patients during the period examined. The most commonly acquired Hb variant was Hemoglobin C (HbC) (64/66 occurrences). More than half of the patients (19/30) acquired HbC on multiple occasions (2-6 times). One patient acquired HbJ and another patient acquired HbD/G in addition to HbC. The segments from donor units were available in some of these cases and hemoglobin electrophoresis (HBE) was performed to confirm the presence of the variant Hb in the donor segments corresponding to that seen on the post-RBCEx sample. CONCLUSIONS: Heterozygous donors are asymptomatic and show no abnormalities during donor screening. Since HBE is not routinely performed on the donor specimen, it may go unrecognized until the post-transfusion recipient results pose diagnostic difficulties. There are no definitive guidelines on deferring these donors; hence one should be cognizant of these findings to prevent misdiagnosis. In a population where HbS negative blood is routinely requested, the effect of other Hb variants remains unknown. None of the patients in our study showed any adverse events due to the acquired Hb variants; however, this is of special concern in the pediatric population where a single RBC unit can contribute a significant portion of the exchanged blood volume. Additionally, donor centers may need mechanisms to confirm the findings and counsel the donors as needed.

A Mosaic Expression of a Hb J-Amiens (HBB: c.54G > T; p.Lys18Asn) and its Interference with Hb A1c Analysis.

We report the case of a 56-year-old Caucasian woman in whom hemoglobinopathy screening was triggered following an aberrant Hb A1c analysis. Preliminary diagnosis of the hemoglobin (Hb) variant was obtained through cation exchange high performance liquid chromatography (HPLC) and gel electrophoresis. DNA analysis confirmed the presence of Hb J-Amiens [ß17(A14)Lys→Asn; HBB: c.[54G > C or 54G > T)]. However, an unbalanced ratio between wild type and mutant signal after direct sequencing and a lower than expected percentage of this Hb variant led to the suggestion of a mosaic expression. Furthermore, different methods [capillary zone electrophoresis (CZE), cation exchange HPLC and boronate affinity] were tested to study the possible interference of this variant with Hb A1c measurements. These investigations showed a clinically relevant difference between the methods tested. Hb A1c analysis may lead to the discovery of new Hb variants or mosaicism for previously described Hb variants. This may have genetic consequences for the offspring of carriers and brings about the question of partner testing.

[Association between hemoglobin Groene Hart and hemoglobin J-Paris-I: first case in Spain]. / Asociación de la hemoglobina Groene Hart con la hemoglobina J-París-I: primer caso en España.

BACKGROUND AND OBJECTIVE: Thalassemias are the most frequent monogenic disorder around the world. α-thalassemias are due to a deficiency of synthesis in the alpha-globin chain of the hemoglobin (Hb). Hb Groene Hart is a hyperunstable variant. In this work, we have studied 24 cases affected by Hb Groene Hart, one of them associated with Hb J-Paris-I. PATIENTS AND METHODS: Twenty-four patients from 17 unrelated families were included in this study. The characterization was done by sequencing. RESULTS: α1 gene sequencing showed the mutation CCT→TCT (Pro→Ser) at codon 119 (Hb Groene Hart) in all patients. In one case, there was an association with Hb J-Paris-I. CONCLUSIONS: In the Hb Groene Hart, the residue 119 of alpha-globin chain is affected. This amino acid has a key role in preserving the stability of alpha-globin chain. It is also remarkable the presence of this variant in both the immigrant and native population. Thus, the identification of Hb Groene Hart carriers should be considered in the screening of α-thalassemia in Spain, as it is done in Northern Africa.

[Analysis of clinical phenotypes of compound heterozygotes of Hb J-Bangkok and ß-thalassemia].

OBJECTIVE: To analyze hematological characteristics of compound heterozygotes of Hb J-Bangkok and ß-thalassemia, and to explore the influence of Hb J-Bangkok on the phenotype of ß-thalassemia. METHODS: Peripheral blood samples from a patient carrying Hb J-Bangkok and a ß-thalassemia mutation, her family members and three sporadic Hb J-Bangkok carriers were collected. RBC analysis and hemoglobin electrophoresis were performed. Genotypes of α- and ß-globin genes were analyzed. RESULTS: The father of the proband and the three sporadic cases were single carriers of Hb J-Bangkok. All of them were asymptomatic and have normal hematological parameters except for an abnormal hemoglobin band detected on hemoglobin electrophoresis. The proband was a compound heterozygote for Hb J-Bangkok and ß-thalassemia mutation IVS-Ⅱ-654. She presented typical ß-thalassemia trait, featuring hypochromic microcytic anemia and increased Hb A2 level. An abnormal hemoglobin band was also detected. CONCLUSION: Carriers of Hb J-Bangkok alone are asymptomatic. Co-existence of Hb J-Bangkok and ß-thalassemia may not aggravate the phenotype. Therefore, couples with one carrying Hb J-Bangkok and another carrying a ß-thalassemia mutation do not require prenatal diagnosis.

Report of haemoglobin J-Toronto and alpha thalassemia in a family from North of Iran.

We report of an Iranian family with history of a rare haemoglobin variant, Haemoglobin J associated with alpha thalassemia, discovered while performing premarital thalassemia screening. In the present study we report the first case of haemoglobin J-Toronto [alpha 5 (A3) Ala > Asp] on -globin gene, found in a 16-year-old female from Mazandaran Province, North of Iran. Further investigation characterized the same mutation for mother and brother of the proband, whilst mother was also a carrier of an alpha thalassemia gene mutation (-alpha3.7). Haemoglobin J-Toronto was previously just reported from Canada and has not been found in any part of Iran.

Isolated Hb Providence ß82Asn and ß82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the ß-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or ß-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two ß-subunit variants with single amino acid mutations at ßLys82→Asp (ßK82D) and at ßLys82→Asn (ßK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its ß-mutant fractions with HbAo. Relative to HbAo, both ßK82N and ßK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of ß-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that ßCys93 and ßCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in ßK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.