Screening of HbE by the New Screening Test: Alcohol HbE Test (AET).

BACKGROUND: Hemoglobin E (HbE) is common in Thailand and the Indochina subcontinent. Implementation of a simple assay to screen for HbE is crucial for use in reproductive counseling, as HbE/ß-thalassemia is a condition with high morbidity and mortality. METHODS: This report developed the alcohol-based HbE screening test named the Alcohol HbE Test (AET). This test was composed of 19% isopropanol in 0.005% Triton-X-100 aliquoted as a 500-µL volume in a 1.5 mL transparent microcentrifuge tube. In performing the AET, 50 µL of EDTA blood was added into the tube, incubated at 37 °C for 30 min before watching for precipitation by eye, which was seen for HbE carriers, not in normal. The test was validated, and assessed for the effect of hematocrit, stability of blood samples, stability of the AET reagent, and interference of HbH. RESULTS: The AET was effective, simple, and less costly than the conventional dichlorophenol indophenol precipitation (DCIP) test. Hematocrit had no effect on the AET results. EDTA blood samples kept in cold conditions (4 °C) were stable for at least 30 days, but only for 2 days at room temperature (30 °C). The aliquoted AET reagent was stable for 30 days in both conditions, while the unaliquoted AET reagent was stable for 6 months. Finally, HbH showed false positive results for the AET. CONCLUSIONS: The AET was successfully developed, and can be an alternative screening test for HbE carriers.

Detecting HbE Gene Using DNA Extracted from Urine Sediments by Chelex-plus-Heating Technique.

Detecting ßE-allele or hemoglobin E (HbE) gene by PCR generally uses DNA prepared from blood leukocytes. However, blood drawing is invasive and prone to injury and infection. The so-called Chelex-plus-heating protocol for DNA extraction from urine sediments was performed in this study. In this protocol, urine sediments were incubated at 37°C with Chelex-100 resin, followed by heating in boiling water for 20 min, and were spun for 1 min to harvest the DNA-containing supernatant. The obtained DNA was subsequently used in amplification refractory mutation system (ARMS)-PCR for detecting ßE-allele. The ARMS-PCR results obtained from urine-DNA were compared to those produced by ARMS-PCR using blood-leukocyte DNA. It was found that the Chelex-plus-heating technique successfully released DNA of good quality with sufficient quantity from urine sediments. Twenty microliters of urine having ∼1111 cells/ml was sufficient to provide good-quality DNA for PCR reaction for HbE genotyping by ARMS-PCR. It was concluded that the Chelex-plus-heating technique was suitable for preparing the DNA from urine sediments. Being simple and less costly, this technique should promote effective control of HbE for countries having a limited budget.

Wide range of F cell levels in healthy Thai adults: Influence of Swiss-type hereditary persistence of foetal haemoglobin & ß-haemoglobinopathy.

Background & objectives: Swiss-type hereditary persistence of foetal haemoglobin (HPFH) has been shown to be responsible for the wide range of F cell levels in healthy Thai adults. However, a survey for F cells in healthy Thai adults has not been performed. This study was conducted to determine the F cell distribution in adult Thai blood donors and to assess the possible involvement of ß-thalassaemia and haemoglobin E (HbE) carriers in increased HbF levels. Methods: Thai blood donors (n=375, 205 males and 170 females) were included in the study. Blood samples were collected for measuring haemoglobin (Hb) concentration and haematocrit (Hct) and F cell levels. Hb and Hct levels were determined by automated blood counter, while F cells were quantified by flow cytometric analysis of F cells stained by fluorescein isothiocyanate-conjugated anti γ-globin monoclonal antibody. Finally, F cell levels were compared between blood samples having mean corpuscular volume (MCV ) <80 fl and ≥80 fl as well as between ß-haemoglobinopathies (HbE and ß-thalassaemia carriers) and normal adults. Results: F cell levels varied markedly spanning 0.80-39.2 per cent with a positively skewed distribution. Thirty two per cent of these individuals had F cell levels more than the 4.5 per cent cut-off point. F cell levels in females were significantly higher than those in males (P<0.05). F cell levels in individuals having MCV <80 fl were significantly higher than those having MCV ≥80 fl (P<0.05). ß-haemoglobinopathy (HbE and ß-thalassaemia carriers) had significantly higher F cell levels than normal individuals (P<0.05). Interpretation & conclusions: The present results showed that besides Swiss-type HPFH, the ß-haemoglobinopathy was expected to be involved in increased F cell levels in adult Thais. Thus, influence of ß-haemoglobinopathy must be considered in interpreting F cell levels in area endemic of this globin disorder.

The in-house monoclonal antibody against γ-globin chain; Thal N/B, accurately measured F cells in SEA-α thalassemia 1 trait.

The monoclonal antibody (mAb) against γ-globin chain clone Thal N/B was produced. It was aimed at measuring the quantity of HbF (α2γ2)-containing red blood cells or F cells by flow cytometry. However, it may cross-react with Hb Bart's (γ4) which is present in the SEA-α thalassemia 1 trait. We measured FC levels by flow cytometry using this in-house mAb for 100 blood samples. Prevalence of high FC trait in this cohort was 51%. Ten of 12 SEA-α thalassemia 1 trait were included in the 51% high FC individuals. Comparing FC levels in the 51 high FC individuals having and not having the SEA-α thalassemia 1 trait showed no difference of FC levels. It was concluded that Hb Bart's did not interfere with FC measurement by flow cytometry using the in-house Thal N/B mAb. Therefore, it can be used for measuring F cell levels in regions having a high prevalence of the SEA-α thalassemia 1 trait. The findings of this research should apply to other clones of anti-γ-globin chain mAb that are aimed for HbF/FC quantification.

A Simple Whole-Blood Polymerase Chain Reaction without DNA Extraction for Thalassemia Diagnosis.

Polymerase chain reaction (PCR) diagnosis of thalassemia usually relies on using genomic DNA. Preparing the genomic DNA can lead to sample-to-sample contamination. This report was aimed to establish the PCR protocol using whole-blood for detecting mutations of α- and ß-globin genes causing the thalassemia syndrome. First, the PCR facilitators, betaine and bovine serum albumin (BSA), were tested, simultaneously with an adjustment of PCR thermal cycler and of whole-blood volume. Thereafter, the established whole-blood PCR was applied for detecting, in both known and unknown samples, the HBA1 Southeast Asian (- -SEA) (NG_000006.1: g.26264_45564del19301) deletion, Hb Constant Spring (Hb CS, HBA2: c.427T>C, αCSα), codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) deletion, -28 (A>G) (HBB: c.-78A>G) and codon 26 (G>A) (Hb E or HBB: c.79G>A). It was shown that the whole-blood PCR worked successfully in 9.0% (w/v) betaine, with 1 µL of EDTA whole blood and with addition of 10 heat-cool steps (3 min. at 94 °C, followed by 3 min. at 55 °C) prior to the typical thermal cycles for the mutations. The capability of the new whole-blood PCR was similar to that of the typical DNA-based PCR. Therefore, the newly established whole-blood PCR could be performed for PCR diagnosis of thalassemia. Using this platform, sample-to-sample contamination should be eliminated.

A modified sandwich ELISA for accurate measurement of HbF in α-thalassemia carriers containing Hb Bart's and Hb Portland 1.

Hemoglobin F (HbF) in blood lysate can be accurately measured by various methods, including immunoassay. In this study, we have produced polyclonal antibody (pAb) against HbF and established a modified sandwich-type ELISA for HbF quantification in blood lysates. The modified sandwich ELISA utilized anti-γ-globin monoclonal antibody clones Thal N/B as the capture antibody (Ab) coated on solid-phase, fluorescein isothiocyanate (FITC)-labeled pAb as the detecting Ab, and HPR-labeled anti-FITC Ab as the signal-generating Ab. By using an optimized blood lysate dilution, the HbF could be measured with no interference from hemoglobin Bart's (Hb Bart's) and hemoglobin Portland (Hb Portland 1) presented in α-thalassemia carriers. HbF levels measured by the modified sandwich ELISA were comparable to those quantified by the standard cation-exchange high performance liquid chromatography. We suggested that this modified sandwich ELISA was able to accurately measure HbF levels even in α-thalassemia carriers containing Hb Bart's and Hb Portland 1 and be an alternative method for HbF measurement.

Hemolysis area: A new parameter of erythrocyte osmotic fragility for screening of thalassemia trait.

BACKGROUND: One-tube osmotic fragility test (OFT) is widely used for screening thalassemia traits. Interobserver variation may occur with 0.36% NaCl-based OFT due to the naked eye result reading style. PURPOSE: The purpose of this study was to establish and evaluate the novel numerical OFT-based parameter, so-called hemolysis area (HA), in screening thalassemia traits. MATERIALS AND METHODS: The portable spectrophotometer was invented capable of calculating the HA values. The HA values were then compared among 69, 156, and 19 blood samples having positive, negative, and suspicious 0.36% NaCl-based OFT results, respectively; 109 and 135 blood samples having mean corpuscular volume (MCV) ≤80 fL and >80 fL, respectively; and 138 and 106 blood samples having mean corpuscular hemoglobin (MCH) ≤27 pg and >27 pg, respectively. In addition, the HA values were compared in 166 blood samples having different globin gene genotypes. Finally, the HA cutoff value was determined by receiver operation curve (ROC) analysis. RESULTS: The HA values in samples having positive, suspicious, and negative 0.36% NaCl-based OFT were 33.3 ± 14.4, 42.9 ± 10.5, and 65.3 ± 13.4, respectively; in sample having MCV ≤80 fL and >80 fL were 43.1 ± 19.6 and 63.8 ± 14.5, respectively; and in samples having MCH ≤27 pg and >27 pg were 46.7 ± 20.1 and 64.8 ± 14.2, respectively. The HA values in normal, hemoglobin E, SEA-α thalassemia 1, and ß-thalassemia traits were 67.1 ± 12.6, 36.4 ± 13.9, 20.2 ± 4.8, and 18.6 ± 1.1, respectively. All were significantly different. ROC analysis established 52.4 as the HA cutoff that had comparable effectiveness to the conventional screening tests. CONCLUSION: The new HA value was effective and could be an alternative choice for screening thalassemia traits.

The Hb E (HBB: c.79G>A), Mean Corpuscular Volume, Mean Corpuscular Hemoglobin Cutoff Points in Double Heterozygous Hb E/- -SEA α-Thalassemia-1 Carriers are Dependent on Hemoglobin Levels.

Identifying double heterozygosities in Hb E (HBB: c.79 G>A)/- -SEA (Southeast Asian) (α-thalassemia-1) (α-thal-1) in patients first diagnosed as carrying Hb E is important in thalassemia control. Low Hb E, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) levels have been observed in this double heterozygosity. However, the cutoff points of these parameters have never been systematically established. Here, we analyzed Hb E and red blood cell (RBC) parameters in 372 Hb E patients grouped by Hb levels, by the status of - -SEA and -α3.7 (α-thal-2; rightward) deletions, to establish the cutoff points. Then, the established cutoff points were evaluated in 184 Hb E patients. It was found that the cutoff points of Hb E, MCV, MCH were significantly dependent on the Hb levels. In the group having Hb levels <10.0 g/dL, the cutoff points of Hb E, MCV and MCH were 21.2%, 64.9 fL and 21.0 pg, respectively, and were 25.6%, 72.8 fL and 23.9 pg, respectively, in the group having Hb levels 10.0-11.9 g/dL. Finally, in the group having Hb levels ≥12.0 g/dL, the cutoff points of Hb E, MCV and MCH were 27.1%, 76.7 fL and 25.3 pg, respectively. Thus, to screen for the double heterozygous Hb E/- -SEA anomaly in patients initially diagnosed as carrying Hb E, the Hb levels must be taken into account in choosing the suitable cutoff points of these three parameters.

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

The associations of SEA-alpha thalassemia 1, XmnI-Ggamma polymorphism and beta-globin gene mutations with the clinical severity of beta-thalassemia syndrome in northern Thailand.

BACKGROUND: At least three genetic factors including beta-thalassemia mutations, alpha-thalassemia, and XmnI-Ggamma polymorphism were shown to modify clinical symptoms in beta-thalassemia disease. OBJECTIVE: To determine associations of beta-thalassemia mutations, SEA-alpha thalassemia 1, and XmnI-Ggamma polymorphism, and clinical severity of beta-thalassemia in northern Thailand. MATERIAL AND METHOD: Thirty-two beta-thalassemia major and 28 beta-thalassemia intermedia attending the Thalassemia Clinic at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand were recruited The beta-globin gene mutations and SEA-alpha thalassemia 1 were determined by MS-PCR and Gap-PCR, respectively. The XmnI-Ggamma polymorphism was identified by RFLP analysis. Odds ratio was calculated to evaluate the associations of these three genetic factors and clinical symptoms. RESULTS: Eight beta-globin gene mutations (both beta0 and beta+) were found Twenty-nine point one percent of the patients had at least one XmnI-Ggamma site (XmnI-Ggamma: +) and 4.1% of the patients were heterozygote for the SEA-alpha thalassemia 1. The beta-globin gene mutations showed maximal impact and the XmnI-Ggamma polymorphism had minimal influence on clinical severity in this cohort. The SEA-alpha thalassemia 1 had the least effect on the clinical severity due to its low prevalence in these patients. CONCLUSION: Although these three genetic factors play roles in modifying clinical symptoms of beta-thalassemia, the beta-thalassemia mutations should be considered first, followed respectively by the XmnI-Ggamma polymorphism and the SEA-alpha thalassemia 1, in management and prenatal diagnosis of beta-thalassemia in northern Thailand.

Effect of Swiss-type heterocellular HPFH from XmnI-Gγ and HBBP1 polymorphisms on HbF, HbE, MCV and MCH levels in Thai HbE carriers.

Relationships of Swiss-type heterocellular HPFH as functions of XmnI-(G)γ and HBBP1:rs2071348 polymorphisms and HbF, HbE, MCV and MCH in HbE carriers were evaluated in 52 non-anemic and α-thalassemia-free Thai HbE carriers. HbF and HbE levels were measured using cation-exchange HPLC. MCV and MCH were determined using an automated blood counter. The XmnI-(G)γ polymorphism was identified by XmnI digestion of amplified products, and the HBBP1:rs2071348 polymorphism by tetra-ARMS-PCR. HbF levels in HbE carriers were higher than those in normal individuals. HbF levels >0.8 % indicated the Swiss-type heterocellular HPFH in these subjects, rendering a prevalence of 40.4 %. The XmnI-(G)γ (+) and HBBP1:rs2071348 (C) alleles were modestly positively correlated with elevated HbF, elevated MCH and lowered HbE values. This study thus confirms the influence of the XmnI-(G)γ and HBBP1:rs2071348 polymorphisms on HbF production. The present study demonstrates the association of XmnI-(G)γ and HBBP1:rs2071348 with HbF, HbE, MCV and MCH in HbE carriers for the first time, and highlights the effect of elevated HbF production on HbE levels.

A simple and highly sensitive ELISA for screening of the α-thalassemia-1 Southeast Asian-type deletion.

Couples in which both partners carry the α-thalassemia-1 trait have a 25% risk of hemoglobin Bart's hydrops fetalis in each pregnancy. Identification of α-thalassemia-1 trait is, therefore, necessary in order to control this severe form of α-thalassemia. We have generated monoclonal antibodies specific to the ζ-globin chain without cross reaction with other globin chains. A simple and sensitive ELISA was developed by using poly-l-lysine to increase the protein binding to the ELISA plate. The developed poly-l-lysine pre-coated ELISA has a very high sensitivity (100%) and specificity (97%) for detection of carriers of α-thalassemia-1 with Southeast Asian-type deletion.

Screening for co-existence of α-thalassemia in ß-thalassemia and in HbE heterozygotes via an enzyme-linked immunosorbent assay for Hb Bart's and embryonic ζ-globin chain.

We sought to demonstrate the ability of levels of Hb Bart's and ζ-globin chain quantified by enzyme-linked immunosorbent assay (ELISA) in detecting α-thalassemia in ß-thalassemia and HbE heterozygotes. We developed an in-house sandwich ELISA method using monoclonal antibodies (mAbs) to Hb Bart's and ζ-globin chain, and quantified levels of Hb Bart's and ζ-globin chain in 172 and 223 anonymous blood samples of ß-thalassemia and HbE heterozygotes, respectively. Genotypes of α-thalassemia 1, ß-thalassemia were identified, and HbE allele was confirmed using a newly developed multiplex allele-specific PCR. The in-house sandwich ELISA method detected Hb Bart's in 6.4% of ß-thalassemia heterozygotes, of which 5.2% showed detectable amounts of the ζ-globin chain. 15.2% of individuals heterozygous for HbE showed a detectable amount of Hb Bart's, and the ζ-globin chain was detected in 11.2% of this cohort. All samples having detectable amounts of Hb Bart's and the ζ-globin chain were verified to be SEA-type α-thalassemia 1. ELISA-quantified Hb Bart's and ζ-globin chain levels can be used to detect double heterozygosity of α- and ß-thalassemia and of α-thalassemia and HbE. This strategy may be useful in screening for co-existence of α-thalassemia in ß-thalassemia and in HbE heterozygotes, particularly in countries where α-, ß-thalassemia and HbE are endemic.

Sandwich ELISA for hemoglobin A2 quantification and identification of beta-thalassemia carriers.

Hemoglobin (Hb) A2 (alpha2delta2) is a minor hemoglobin in human red blood cells. An abnormal increase in the level of HbA2 is the most significant parameter in the diagnosis of beta-thalassemia carriers. In this study, we produced two monoclonal antibodies (mAbs) that specifically react to the delta-globin chain of HbA2. A sandwich type ELISA was developed employing the produced anti-HbA2 mAbs. HbA2 levels quantified by the developed sandwich ELISA were highly correlated with those obtained from the standard HPLC method (r = 0.934, p < 0.001). HbA2 levels determined by the ELISA were 4.4 +/- 1.9% in beta-thalassemia heterozygotes compared to 1.4 +/- 0.8, 1.9 +/- 0.8, 1.5 +/- 0.8 and 1.5 +/- 0.6% in normal subjects, HbE heterozygotes, suspected alpha-thalassemia heterozygotes and HbE homozygotes, respectively. Using a cut-off value of 2.5%, beta-thalassemia heterozygotes could be separated from non-beta-thalassemia heterozygotes with the same accuracy as obtained using the standard HPLC method. More importantly, the developed ELISA was able to determine HbA2 levels in HbE-bearing individuals which could not be done by the HPLC method. Our results suggest that this sandwich ELISA can be applied for mass screening for beta-thalassemia heterozygotes, especially in resource-limited countries, where beta-thalassemia is highly prevalent.

Simple method for screening of alpha-thalassaemia 1 carriers.

Alpha-thalassaemia 1 genetic disorder occurs when there is a deletion of two linked alpha-globin genes. The interaction between these abnormal genes leads to the most severe type of thalassaemia disease, haemoglobin (Hb) Bart's hydrops fetalis. The identification of alpha-thalassaemia 1 carriers and genetic counselling are essential for the prevention and control of severe thalassaemia diseases. In this study, we have developed a rapid screening method for identifying alpha-thalassaemia 1. A sandwich-type immunochromatographic (IC) strip test was developed, using the generated monoclonal anti-Hb Bart's antibody, to trace the Hb Bart's in haemolysates. When assayed by our IC strip test, all alpha-thalassaemia 1, HbH disease, HbH-Constant Spring (H-CS) disease, HbH-CS and heterozygous HbE (CSEA) Bart's disease, and homozygous alpha-thalassaemia 2 showed positive results. No false negative results were observed in these blood samples. In alpha-thalassaemia 2 heterozygotes, 83% of them showed positive reactivity. Among HbE (both homozygotes and heterozygotes), beta-thalassaemia (heterozygotes, homozygotes and beta-thalassaemia/HbE) and normal subjects, the IC strip test revealed negative reactivity of 100, 85 and 97%, respectively. These results indicate that this novel immunodiagnostic kit, in combination with red blood cell indices, is suitable for screening and ruling out mass populations for the presence of alpha-thalassaemia 1.

Perinatal zidovudine prophylaxis in HIV type-1-infected pregnant women with thalassaemia carriage in Thailand.

BACKGROUND: To investigate a possible interaction between alpha-thalassaemia, beta-thalassaemia and haemoglobin-E trait and the haematological parameters of HIV type-1 (HIV-1)-infected pregnant women receiving zidovudine prophylaxis for the prevention of mother-to-child HIV-1 transmission in Thailand. METHODS: The study sample was composed of HIV-1-infected pregnant women receiving zidovudine (300 mg twice daily) from 28 weeks of gestational age to delivery as part of the Perinatal HIV Prevention Trial (PHPT-1), a large trial investigating zidovudine use in pregnancy. These women were randomly selected and screened for haemoglobin abnormalities. Haemoglobin levels, haematocrit and erythrocyte, leukocyte, absolute neutrophil and absolute lymphocyte counts were measured at 26, 32 and 35 weeks of gestation and at delivery. PCR genotyping techniques were used to screen for haemoglobin abnormalities, which included alpha-thalassaemia-1 Southeast Asian type deletion, beta-thalassaemia mutation (codons 41/42 [-TCTT], codon 17 [A-->T], intervening sequence-I nucleotide 1 [G-->T], codons 71/72 [+A]) and haemoglobin-E trait. The evolution of haematological parameters between 26 weeks and delivery was compared according to thalassaemia carriage using linear mixed models adjusted for baseline sociodemographic characteristics, HIV clinical stage, CD4+ T-cell count and viral load. RESULTS: At baseline, women with thalassaemia or haemoglobin-E trait had significantly lower haemoglobin level and red blood cell counts than women with no haemoglobin abnormalities, whereas absolute neutrophil and leukocyte counts were significantly higher. Exposure to zidovudine until delivery did not increase this difference. CONCLUSIONS: Zidovudine exposure did not appear to have increased haematological toxicity in HIV-1-infected pregnant women with thalassaemia.

Evidence of genetic interaction between the beta-globin complex and chromosome 8q in the expression of fetal hemoglobin.

During human development, the switch from fetal to adult hemoglobin (Hb) is not complete with the residual gamma-globin expression being restricted to a subset of erythrocytes termed "F cells" (FC). Statistical analyses have shown the FC trait to be influenced by a common sequence variant (C-->T) at position -158 upstream of the Ggamma-globin gene, termed the "XmnI-Ggamma polymorphism." The XmnI-Ggamma site is believed to be involved in the expression of the Ggamma-globin gene through interaction with transcription factors, and polymorphisms in the transcription factors could be influencing fetal Hb expression, conditional on the XmnI-Ggamma site. Using a two-locus model, in which the second locus was the known quantitative-trait locus (QTL) at the XmnI-Ggamma site, we showed suggestive linkage to chromosome 8q. A maximum single-point LOD score of 4.33 and a multipoint LOD score of 4.75 were found in a 15-20 cM region of chromosome 8q. A single-locus analysis failed to show linkage of FC to the region when the XmnI-Ggamma site was accounted for by removing its effects from the data or including it as a covariate. Results of the single-locus analysis were significant when the effects of the XmnI-Ggamma site were not accounted for in any way. The results of analysis in a large Indian kindred indicate that there is an interaction between the XmnI-Ggamma site and a QTL on chromosome 8q that is influencing the production of fetal Hb.