Comparison of fast protein liquid chromatography (FPLC) with HPLC, electrophoresis & microcolumn chromatography techniques for the diagnosis of β-thalassaemia.

Background & objectives: β-thalassaemia is a genetic disorder and an important health problem around the world. Quantitative haemoglobin A2 (HbA2) levels are used for the diagnosis of β-thalassaemia. The conventional methods are high performance liquid chromatography (HPLC), electrophoresis, and microcolumn chromatography techniques. We established a fast protein liquid chromatography (FPLC) method, to measure quantitatively of HbA2 levels, and compared its efficacy with conventional methods. Methods: The FPLC method, using a DEAE Sepharose, Hi Trap anion-exchange column chromatography technique was set up for HbA2 measurement. In this study, 220 blood samples were screened for haemoglobin type by FPLC technique and also using HPLC, microcolumn chromatography and electrophoresis. Results: The FPLC results were highly correlated (r = 0.985, P<0.001) with those of HPLC for quantification of HbA2 as well as cellulose acetate electrophoresis (r = 0.977) and microcolumn chromatography (r = 0.980). The FPLC method showed 100 per cent sensitivity and specificity, positive and negative predictive value for β-thalassaemia diagnosis. In addition, the FPLC method was simple, rapid, low cost and reproducible. The HbA2/E range of FPLC for β-thalassaemia was 6-10 per cent, HbE trait was 10-40 per cent, β-thalassaemia/HbE was 40-60 per cent and homozygous HbE was more than 60 per cent. Interpretation & conclusions: Our findings suggested that FPLC method could be used as a cost-effective method for routine β-thalassaemia diagnosis.

Hemoglobin E detection using PCR with confronting two-pair primers.

OBJECTIVES: To develop and apply the polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for detection and identification of hemoglobin E (Hb E). MATERIAL AND METHOD: Fifty unrelated northern Thais were included in the present study. DNA was extracted from peripheral blood mononuclear cells and targeted to amplify by PCR-CTPP. The amplified product was analyzed and compared with the reference hemoglobin electrophoresis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: The results validated a completely concordant among these three methods consisting of 74%, 24%, and 2% identified as normal, heterozygous, and homozygous Hb E type, respectively. CONCLUSION: Successful Hb E genotyping by PCR-CTPP was introduced. It allows for confirming and simultaneously detection with other thalassemia mutations.

Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E.

OBJECTIVE: To evaluate hemoglobin E screening tests in a large scale of cases. MATERIAL AND METHOD: A cross-sectional descriptive study was conducted Whole blood obtained from subjects was evaluated for CBC, OF, DCIP, and hemoglobin typing. RESULTS: Five hundred twenty seven hemoglobin E and 280 reference subjects participated. DCIP's sensitivity, specificity, positive predictive value, and negative predictive value were 97.16%, 98.93%, 99.42%, and 95.19%, respectively. These values of OF were 69.12%, 80.00%, 86.67%, and 57.88%, respectively. In the combination of DCIP and OF gave rise to these values of 99.43%, 79.29%, 90.03%, and 96.67%, respectively. Finally the combination of DCIP and MCV < 80 fL resulted in these values to be 99.43%, 98.93%, 99.43%, and 98.93%, respectively. False positive and false negative rate were 1.07% and 0.57%, respectively. CONCLUSION: Combination of DCIP and MCVwas better than that of DCIP and OF in hemoglobin E screening.

Validation of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E.

The strategy for screening of thalassemia and Hb E by a combination of osmotic fragility (OF) test and dichlorophenol indophenol precipitation (DCIP) test was validated with 436 unrelated Thai subjects. Hemoglobin (Hb) typing, Hb A2 quantitation, PCR and DNA sequence analysis were used as confirmatory methods for diagnosis of thalassemia and hemoglobinopathy. The sensitivity and specificity of this strategy was 100% and 79.7%, respectively. The results assessed by two medical scientists were exactly the same with 93.3% accuracy in comparison with the confirmatory methods. A combination of OF and DCIP has been shown to be a reliable, rapid, simple and sensitive strategy for screening thalassemia and Hb E in the Thai population.

Antioxidant in plasma of hemoglobin-E trait.

A study of antioxidant levels among Thai subjects with a hemoglobin E trait was undertaken. The objective of this study was to determine whether the antioxidant level would be disturbed in the HbE condition. All 185 volunteer subjects, 171 normal healthy subjects and 14 HbE carriers were recruited. For each case, an antioxidant determination was performed using the Trolox equivalent antioxidant capacity (TEAC) method. According to this study, the average antioxidant level in the healthy group was 3.439 +/- 0.220 mM Trolox equivalent, and in HbE trait group was 3.276 +/- 0.209 mM Trolox equivalent. There was a significant decrease of the antioxidant level in the HbE trait group (p = 0.008).

Hemoglobin E-beta thalassemia: factors affecting phenotype.

The phenotype of E-beta-thalassemia is affected by several genetic factors. The aim of this study was to analyze severity of E-beta-thalassemia and correlate with HbE, HbF, E/F ratios, beta-mutation and Xmn I polymorphism. Thirty cases of E-beta-thalassemia (23 with childhood onset) were studied. HbE levels were quantitated by HPLC. Xmn1 polymorphism and beta-mutations were studied by PCR-RFLP and ARMS respectively. Commonest features were pallor (100%), splenomegaly (74%), and hepatomegaly (65%), 43% (10/23) were on regular transfusions at diagnosis. One case presented with paraplegia. Patients heterozygous for Xmn I polymorphism (+/-) had later onset (>3 yrs) compared to homozygous (-/-) absence (0.5-2.8 yrs). Most (69.6%) showed beta-mutation IVS 1-5 (G-->C). Negative correlation was found between age of onset and HbE. Thus, presentation is similar to previously reported Thai cases. Heterozygosity of Xmn I polymorphism also delays disease onset. Early diagnosis facilitates appropriate management and prenatal diagnosis.

Hemoglobin E levels in double heterozygotes of hemoglobin E and SEA-type alpha-thalassemia.

Coinheritance of alpha-thalassemia and hemoglobin E (Hb E) is prevalent in Thailand, where the gene frequencies of thalassemia and hemoglobinopathies are high. Hb E carriers with, concomitant inheritance of alpha-thalassemia 1 are known to have a lower level of Hb E. In this study, we reviewed the Hb E levels in Hb E carriers, who either had or did not have Southeast Asian (SEA)-type alpha-thalassemia, in order to seek a Hb E level that may be used as a predictor for concomitant alpha-thalassemia carrier status. The Hb E levels as measured by microcolumn chromatography in 844 Hb E carriers detected during a prenatal screening program for severe thalassemia at Chiang Mai University Hospital were reviewed. Hb E levels ranged from 12.3-35.0% (23.3 +/- 3.1%) in 751 Hb E carriers without SEA-type alpha-thalassemia and from 11.6-32.0% (17.0 +/- 3.7%) in 93 concomitant Hb E and SEA-type alpha-thalassemia carriers. The difference was significant (p < 0.01). However, the absence of SEA-type alpha-thalassemia could not be predicted by the higher Hb E level alone, as 3% of double heterozygotes demonstrated a level of more than 25%. Our study confirms a lower Hb E level in double heterozygotes with Hb E and SEA-type alpha-thalassemia. Nevertheless, the difference does not provide sufficient discriminatory power for the reliable exclusion of alpha-thalassemia status.

Case report post-splenectomy sepsis in thalassemic patients.

Thalassemia is the commonest hemoglobinopathy in Malaysia. Patients with thalassemia major are transfusion dependent, and a large proportion of them will require splenectomy. As this particular group of patients is immunocompromized, overwhelming sepsis is a recognized complication. We report a series of three patients who all developed intra-abdominal abscesses following splenectomy.

Human herpes virus 6 antibodies in beta-thalassemia/hemoglobin E pediatric patients.

Human herpesvirus 6 (HHV-6) is a viral pathogen that causes exanthem subitum in children. It has also been identified as the cause of life-threatening illness in immunocompromised pediatric patients and transplant recipients. We undertook a serological study of HHV-6 IgM and IgG antibody among 29 children (12 females and 17 males) with beta-thalassemia/HbE disease. The rate of infection was 86.2%; the rates of early recent infection (IgM positive only), recent infection (both IgM and IgG positive) and past infection (IgG positive only) were 13.8%, 41.4% and 31.0%, respectively. The geometric means of the IgM and IgG titers of the splenectomy group (9 cases) were 10.15 units and 11.18 units, respectively. The geometric means of the IgM and IgG titers of the non-splenectomy group (20 cases) were 10.10 units and 12.84 units, respectively. According to this study, the prevalence of HHV6 infection among pediatric patients with beta-thalassemia/HbE is very high; morever, the significantly higher titer among these patients may imply a high risk for further possible bone marrow transplantation. Increased awareness of HHV-6 infection among this population is necessary.

Correlation between some discrimination functions and hemoglobin E.

The most widely used discriminant functions and red cell indices for differential diagnosis of thalassemia traits from iron deficiency anemia were evaluated for their abilities to identify HbE-containing blood samples. The functions were as follows: F1 = 0.01 x MCH x (MCV)2; F2 = RDW x MCH x (MCV)2/Hb x 100; F3 = MCV/RBC; and F4 = MCH/RBC. Other red cell parameters including RDW, hemoglobin content, mean cell volume, mean cell hemoglobin as well as red cell counts, were also evaluated to distinguish HbE from the normal population. Hemoglobin electrophoresis was used as a confirmatory test. The results showed that F1, F2 and F3 as well as other red cell parameters of HbE-containing samples were different from those of HbA2A-containing red cells although there was no statistical significance. However, F4 and MCHC showed no difference between the two groups. It can be concluded from the present study that identification of hemoglobin E especially the heterozygous form by using parameters from an electronic cell counter is not easy. Discriminant functions and red cell indicies might be used as an initial diagnosis. But confirmation is needed in all cases. Applying the MCV of 80 fl will miss 5 per cent of hemoglobin E carrier but will not miss the homozygous form.

Preventation of thalassemia in Australia.

Screening for thalassemia and other hemoglobinopathies in the major maternity hospitals in Melbourne, Australia has shown that 6% of the patient population carries a clinically significant genetic abnormality. The most common of these are beta-thalassemia (3%). HbS (1.8%), HbE (0.5%) and alpha0 thalassemia (0.4%). Approximately 60 prenatal diagnoses for the clinically significant combinations of these abnormal genes are performed annually in the 2 major centers of Melbourne and Sydney. The majority of these prenatal diagnoses are for beta-thalassemia major (65%). whilst 11% are for Bart's hydrops fetalis, 8% for HbE/beta-thalassemia. 6% for HbS/beta-thalassemia, 2% for sickle cell anemia and the remaining 8% for other combinations of thalassemia/hemoglobinopathies. Of the 178 patients with beta-thalassemia major, sickle cell disease or beta-thalassemia in combination with HbE or HbS, only 5 are less than 5 years old, reflecting both the success of the screening program and the increasing acceptance by couples of 1st trimester prenatal diagnosis.

Prevalence of HB E from cord blood samples and after one year follow-up.

Hemoglobin (Hb) E is the most prevalent hemoglobinopathy in Southeast Asia. The prevalence of this condition varies from 9-60% of the population in different regions of Thailand and has the highest prevalence the northeast of the country. Neonatal diagnosis of Hb E can be made by detecting the Hb band in cord blood samples at the Hb A2 position using starch gel and cellulose acetate electrophoresis. Our study, performed in Bangkok, in the central part of Thailand, resealed that 182 out of 1,015 cord blood samples (17.9%) contained Hb E in amounts of between 1.9 and 10.0%. The cases who had Hb A, F and E with or without Hb Bart's were initially included in the study. These cases were suspected to have the Hb E trait. One hundred and seven cases (58.89%) were available for follow up and in all of these, Hb E could be detected throughout the study. A sharp increase in the amount of Hb E was observed at the 3 months follow-up appointment. One year follow-up could be made in 72 cases (39.6%) when the percentage of Hb E was around 25%. We conclude that measurement of Hb E in cord blood an easily accessible, simple, practical and sensitive procedure which can be used to study the Hb E hemoglobinopathy which is widely distributed in Thailand and Southeast Asia.

Hemoglobin typing by high performance liquid chromatography.

To evaluate the potential application of high performance liquid chromatography or HPLC in performing hemoglobin typing, comparison between this technique and routine methods was carried out. Blood specimens from Pediatric Hematology Unit, Research Center, Faculty of Medicine, Ramathibodi Hospital were examined by these methods. The level of Hb A2, Hb E and Hb F were compared. Hb A2 level determined by HPLC and electrophoresis was statistically significantly different but correlated well. Good correlation was found between Hb A2 level from HPLC compared with microcolumn chromatography although there was a statistical difference. No difference but very good correlation was found between the level of Hb E from HPLC compared with electrophoresis. Statistical difference was encountered when Hb F level determined by HPLC was compared to that determined by Betke alkaline denaturation test. However, good correlation was observed when the level of Hb F was greater than 2.0%. In conclusion, HPLC could be an alternative way of performing hemoglobin typing provided that Hb F is 10% or more, by calculating from the equation: Alk F = (0.83 x Hb F by HPLC) - 0.98.

Difference in pattern of erythropoietin response between beta-thalassemia/hemoglobin E children and adults.

Thalassemia is one of the most common genetic disorders in Thailand. The thalassemic patients have many pathophysiologic changes secondary to chronic anemia. During these last few years there have been many trials to cure or improve the anemic condition in thalassemia by using various agents, including erythropoietin (EPO). Thus it is very important to understand the EPO response to different degree of anemia in the thalassemic patients. In this study we evaluated the EPO status in 53 beta-thalassemia/HbE patients, from 4-61 years old, by enzyme-linked immunosorbent assay. The results showed that the levels of EPO in beta-thalassemia/HbE patients were much higher than in normal control subjects: mean +/- SE = 527 +/- 183.20 and 3.45 +/- 0.47 mIU/ml respectively. The reverse correlation between the levels of EPO and hematocrit (r = -0.704) was also observed. There was also a tendency to have higher levels of EPO in beta-thal/HbE children than in adults, although this was statistically insignificant. The observed versus predicted levels of EPO (log O/P ratio) showed that most patients had good EPO response to the degree of anemia. However, inappropriate decrease of EPO response was observed in 8/40 adult patients. The EPO levels in these patients were not correlated with any physical or laboratory studies, including kidney function. We thus propose that if EPO is to be considered as one of the alternative treatment to the thalassemic patients, in the future, it may benefit only the patients with low EPO levels.

Prevalence of hemoglobin E, alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok.

Thalassemia hemoglobinopathies and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency are prevalent in Thailand. We studied the prevalence of these disorders from 1,000 cord bloods collected during 14 months period, using EDTA as anticoagulant. Red blood cell G-6-PD quantitative assay was performed in all male subjects. Nine hundred and eighty five specimens were available for hemoglobin (Hb) typing by starch gel electrophoresis. Further evaluation by cellulose acetate electrophoresis and follow up were made in the cases who had Hb E and/or high level of Hb Bart's. It was found that out of 505 males, 61 cases (12.08%) had G-6-PD deficiency. Among 985 cases studied for Hb typing, 61.92% revealed normal Hb type AF while Hb E was present in 18.68% and Hb Bart's designated alpha-thalassemias were present in 25.18% respectively. Of these 985 cases, 18.78% had low Hb Bart's level ie detectable to 8.2% consistent with alpha-thal2, Hb Constant Spring (CS) or alpha-thal1 trait. Ten cases (1.02%) had high levels of Hb Bart's ranging from 16.1-35% without or with Hb CS and E, and further follow-up revealed homozygous Hb CS, Hb A-E-Bart's, Hb H and Hb H with Hb CS disease. The other 53 cases (5.38%) had low level of Hb Bart's with Hb E consistent with alpha-thalassemia trait with Hb E trait. There were 127 cases (12.89%) who had only Hb E trait and 3 cases (0.3%) who had Hb F and E without Hb A initially.

Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family.

Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/-alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q-chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E.

Expression of hemoglobin E in newborn.

Hemoglobin E(HbE) is an abnormal hemoglobin resulted from a point mutation in codon 26 (GAG-AAG) of beta-globin gene. The mutation causes an amino acid substitution (Glu-Lys) and abnormal splicing in exon 1 that reduce the amount of beta E chain synthesis. While in adult, the HbE can easily be diagnosed, its level in newborn is usually underrepresent. In this study, we examined a relationship between genotype of HbE and the amount of HbE in cord blood. 145 Cord blood specimens were analysed by starch gel electrophoresis and the amounts of HbE were determined by microcolumn chromatography. The zygosity of beta E globin gene was determined by the polymerase chain reaction. The levels of HbE were 3.17 +/- 1.79% for 59 heterozygotes, 8.55 +/- 2.52% for 3 homozygotes and 0.48 +/- 0.22% in 83 normal newborns. This result provides useful data for a neonatal screening program and implies that expression of HbE in newborn dependent on a copy number of beta E globin gene in each individual.