Hemoglobin H identification by high-performance liquid chromatography in confirmed hemoglobin H disease.

INTRODUCTION: Among hemoglobin (Hb) H disease cases diagnosed by DNA testing in our hemoglobinopathy laboratory, we have noted instances of unreported Hb H from high-performance liquid chromatography (HPLC) results of referring laboratories. METHODS: To characterize these issues, we identified all cases of genotypic Hb H disease diagnosed in our laboratory. HPLC chromatograms were reviewed to determine the presence and retention time of the Hb H peak. RESULTS: Hemoglobin H was not reported in 24.2% of patients (23 of 95) with genotypic Hb H disease. The characteristic prerun peak of Hb H was present on review of all eight Variant or Variant II ß-thalassemia short-program chromatograms. Elevated Hb F (≥3%) was reported in 14 cases. The Hb H peak was found in the Hb F window in 11 dual program cases. The incorrect identification of Hb H as elevated Hb F resulted in two testing referrals for 'δß-thalassemia'. CONCLUSIONS: Hemoglobin H may go unreported due to failure to examine for or recognize its peak on Variant or Variant II ß-thalassemia short-program chromatograms. Elution of Hb H in the Hb F window resulted in misidentification of Hb H for Hb F and may indicate a Variant II HbA2 /HbA1C program software error. Our findings highlight the need for careful chromatogram inspection and clinical correlation in the diagnosis of Hb H disease.

Genetic heterogeneity of hemoglobin AEBart's disease: a large cohort data from a single referral center in northeast Thailand.

AEBart's disease is a thalassemia intermedia usually characterized by the interaction of α(0)-thalassemia with either deletional or non-deletional α(+)-thalassemia in Hb E heterozygote. Genotypic and phenotypic features are heterogeneous. We studied the hematologic and molecular characteristics of this disease in a cohort of 173 Thai patients encountered at our center in northeast Thailand. Hemoglobin and DNA analyses identified patients with deletional AEBart's disease (n=84), Hb Constant Spring AEBart's disease (n=81), Hb Paksé-AEBart's disease (n=5), AEBart's disease with codon 30 mutation (n=1) and two hitherto un-described forms of AEBart's disease due to interaction of Hb E heterozygote and α(0)-thalassemia with the -α(16.6)kb deletional α(+)-thalassemia (n=1) and Hb Q-Thailand (n=1). Different phenotypic expression of these AEBart's diseases with low Hb, Hct and MCV and increased RDW values with marked reduction in Hb E levels were observed. It was found that all these forms of AEBart's disease showed similar thalassemia intermedia phenotypes but those with non-deletional forms were relatively more anemic. Our data confirm that in such area with high prevalence of hemoglobinopathies such as Southeast Asia, identification of rare thalassemia alleles in a thalassemia intermedia patient should not be ignored. Careful consideration of different phenotypic expression may help in providing presumptive diagnosis of this disease where access to molecular testing is limited. However, molecular diagnostic is useful for predicting the clinical outcome and improving genetic counseling of these complex hemoglobinopathies.

Newborn screening for Hb H disease by determination of Hb Bart's using the Sebia capillary electrophoresis system in southern China.

Hb H (ß4) disease is an inherited hemoglobin (Hb) defect in which three of the four α-globin genes are deleted or dysfunctional. The clinical manifestations vary widely from mild asymptomatic anemia to a severely anemic state. Recent literature suggests that Hb H disease is not as benign a disorder as previously thought. Newborn screening for Hb H disease is especially appealing because the screening test is based on the detection of Hb Bart's (γ4) that is only possible within the newborn period. In a 2-year period of newborn screening, 18 babies were found to have Hb H disease in a total of 9490 newborns. The overall prevalence for Hb H disease among all newborns in southern China is approximately 1 in 500. The correct diagnosis would allow affected infants to be properly cared for and reduce mortality rate.

The Hb H disease genotypes in Southern China.

Abstract We report the genetic data of 435 patients with Hb H (ß4) disease who presented at our center between 2005 and 2012. Our results showed that all patients had the Southeast Asian deletion (- -(SEA)) on one allele. The -α(3.7) (rightward) deletion was the most common on the other allele, followed by the -α(4.2) (leftward) deletion, Hb Constant Spring (Hb CS, α142, Term → Gln; HBA2: c.427T > C) and Hb Quong Sze [Hb QS, α125(H8)Leu → Pro; HBA2: c.377T > C] mutations. Two rare point mutations, α31(B12)Arg → Lys; HBA2: c.95G > A and Hb Zurich Albisrieden [α59(E8)Gly → Arg; HBA1: c.178G > C], were also identified. Four patients had a concomitant ß-thalassemia (ß-thal) heterozygosity. Our results reflect the genetic heterogeneity of Hb H disease and the interaction between Hb H disease and ß-thal trait in Southern China.

Bilirubin peak can be mistaken as Hb Bart's or Hb H on High-performance liquid chromatography.

High-performance liquid chromatography (HPLC) has largely replaced electrophoresis for the identification of hemoglobin (Hb) variants. However, one needs to be vigilant to pick up undue interference such as the presence of a bilirubin peak, which can be mistaken for either Hb Bart's or Hb H on Hb HPLC. Correlation with the clinical context and biochemical parameters, coupled with repeating the run after washing the sample, can resolve this problem. Herein, we report one such case of an acute on chronic liver failure patient who was found to have heterozygous ß-thalassemia (ß-thal) on Hb HPLC with an incidental tall unknown peak that was identified as bilirubin.

Protein conformational heterogeneity as a binding catalyst: ESI-MS study of hemoglobin H formation.

Our previous studies of hemoglobin tetramer assembly in vitro suggested that the initial step in the oligomerization process, which ultimately dictates the high fidelity of the heterotetramer (alpha*beta*)2 assembly, is the binding of a flexible heme-free beta-globin chain to a highly ordered heme-bound alpha*-globin. In this work, we extend these studies to investigate formation of the homotetrameric hemoglobin H, whose formation in vivo is a well-documented clinical consequence of significant overexpression of beta-globin in alpha-thalassemic disorders. Upon reconstitution of the isolated beta-globin with excess heme, the predominant species in the ESI mass spectrum corresponds to the homotetramer beta*4, alongside homodimeric species and monomeric beta-globin chains in both apo and holo forms. The assembly process of the hemoglobin H homotetramer apparently follows a scenario similar to that of a normal heterodimeric hemoglobin (alpha*beta*)2 species, with the asymmetric binding event between compact and flexible polypeptide chains being the initial step. The extreme importance of large-scale chain dynamics and conformational heterogeneity for the protein assembly process is highlighted by the inability of highly structured alpha-globins to undergo ordered oligomerization to form dimers and tetramers as opposed to indiscriminate aggregation.

Effect of HbE and HbH on HbA1C level by ionic exchange HPLC comparing to immunoturbidimetry.

BACKGROUND: The hemoglobin (Hb)A1C level is widely used to monitor diabetes mellitus patients. The N-terminal amino acid valine of its beta chain is glycated. The assay of HbA1C is based on differences in the charge, chemical and structural properties of the protein. METHODS: There are fully automated instruments available in clinical chemistry laboratory to assay HbA1C level. The effect of hemoglobinopathies was studied between an ionic exchange high-pressure liquid chromatography (HPLC) (Bio-Rad Laboratories, USA) and immunoturbidimetry (BM/Hitachi 912 with Roche HBA1CII, Germany-Japan) assay. The influence of high level HbF relative to the HbA1C level by ionic exchange HPLC is known. The effect of HbE and HbH to the HbA1C level by ionic exchange HPLC comparing to immunoturbimetry was examined. The evaluation was performed on 34 normal controls (A2A), 17 beta thalassemia traits (A2 upward arrow A), 36 HbE heterozygotes (EA), 37 HbE homozygotes (EE), 36 beta thalassemia/HbE (EF/EFA), 11 EABart's diseases (EABart's), 34 Hb H diseases (A2/CSAH) and 13 cord blood samples (FA). CONCLUSIONS: Hemoglobinopathies can impact on the assay of HbA1C level such as HbE and HbH to ionic exchange HPLC. Although not studied as yet, this effect may influence the other methods such as affinity chromatography.

Phenotype-genotype correlation in Sicilian patients with Hb H.

We studied 15 Sicilian subjects with Hb H disease correlating clinical examinations with hematological and molecular data. Seven different alpha-tha1 mutations were identified: four deletion types (--MED --CAL, -alpha3.7, -alpha4.2) and three nondeletion types (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). All the patients had a zero-gene chromosome (--MED or --CAL), while the third alpha gene was deleted (-alpha3.7, -alpha4.2) or inactive (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). In patients with the nondeletion genotype the analysis of hematological values revealed lower levels of RBC and Hb A2 and significantly higher levels of Hb H. The clinical variability was remarkable, ranging from totally asymptomatic conditions, casually diagnosed, to severe thalassemia intermedia with marked hemolytic crises, liver and spleen enlargement and the necessity for frequent transfusions. The genotype did not justify the gravity of the phenotype in every case, and the differences in clinical manifestations, also notable, are not easily explainable in subjects who apparently have the same genotype.

Binding of nitric oxide to thiols and hemes in hemoglobin H: implications for alpha-thalassemia and hypertension.

Our earlier studies suggested an association between alpha-thalassemia and hypertension. We postulated that this association might involve trapping of the vasodilator nitric oxide (NO) by hemoglobin (Hb). Hb A has recently been shown to carry NO on its sulfhydryl groups in addition to its hemes. In this report we studied the interaction of purified Hb H as well as Hb A with NO. The number of reactive sulfhydryls were determined spectrophotometrically with bis-dithionitrobenzoate. Spectral studies and nitrosothiol measurements after treatment with NO or nitrosothiols indicated that all eight reactive sulfhydryls of Hb H were capable of binding NO. Hb A, however, was only able to bind and transfer two molecules of NO per tetramer. These findings support the biochemical basis for the association between alpha-thalassemia and hypertension.

Comparison of the HbH inclusion test and a PCR test in routine screening for alpha thalassaemia in Hong Kong.

AIM: To compare the haemoglobin (Hb) H inclusion test with a polymerase chain reaction (PCR) test in routine screening for alpha thalassaemia. METHODS: Ninety nine peripheral blood samples from Chinese patients with mean corpuscular volume below 80 fl were screened for alpha thalassaemia using the HbH inclusion test and by PCR utilising primers bridging the common deletion breakpoint of the South East Asian (--SEA/) deletion. RESULTS: The HbH inclusion test was positive in 78 (79%) patients, 73 (93.7%) of whom carried the (--SEA/) deletion on analysis of their DNA by PCR, as did one patient with a negative HbH inclusion test. CONCLUSIONS: These results suggest that in areas with a high prevalence of the (--SEA/) deletion, such as Hong Kong, the HbH inclusion test can be replaced by PCR as the investigation of choice in screening for alpha thalassaemia.