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S Fucharoen

Publications and source records attributed to S Fucharoen.

At least 55 records · Page 3Linked to original sources
PubMed1999

Standardization on laboratory diagnosis of thalassemia and abnormal hemoglobin.

Thalassemia is one of the most common single gene disorders. The geographic distribution of thalassemia and abnormal hemoglobin has been known for many years. A worldwide significant spread of these abnormal genes, especially from Southeast Asia, occurred in the last two decades. This has resulted in a dramatic increase of Hb E disorders and various Southeast Asian thalassemia genotypes, which means that requests for hemoglobinopathy investigations are likely to increase in many laboratories worldwide. Hemoglobinopathy screening and diagnosis may need to be undertaken antenatally, neonatally and in certain hematological situations. The introduction of automation for hemoglobinopathy screening, including the automated cell counting and HPLC system, is an important advance in technology for hematology laboratories. The instruments need to be calibrated and standardized to get an accurate data for interpretation. Internal and external control samples are also needed. Combination of test results is usually required to achieve a proper diagnosis, which in turn, provide a self-check for each laboratory test.

Adult↗
PubMed1999

Icsh activities and Asia.

The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.

Asia↗
PubMed1999

Detection and structural analysis of abnormal hemoglobins found in Thailand.

In Thailand, some 20 different abnormal hemoglobins have been found in the last 30 years. Most are rare except for Hb E and Hb Constant Spring, found with frequencies of 10-53% and 1-8% respectively in different parts of the country. Most mutations are point mutations, but C-terminal elongations and crossing-over are also found. Most mutations do not cause clinical problems, but some can give rise to mild thalassemia syndromes, or cause problems in association with thalassemia. Abnormal hemoglobins may often be diagnosed by electrophoresis, but some variants have the same mobility, so that other techniques are required, such as HPLC and the use of allele-specific polymerase reaction or oligonucleotide probes. Novel variants, not previously described in Thailand, require structural analysis at the protein and DNA level.

Chromatography, High Pressure Liquid↗
PubMed1998

Membrane phospholipid asymmetry in human thalassemia.

Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe beta-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the alpha- or beta-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of beta-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound alpha-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.

Annexin A5↗
PubMed1998

Detection of zeta-globin chains in the cord blood by ELISA (enzyme-linked immunosorbent assay): rapid screening for alpha-thalassemia 1 (Southeast Asian type).

Fetuses with homozygous alpha-thalassemia 1, in which the deletion of all four alpha-globin genes results in the absence of any alpha-globin chains, are severely anemic with clinical features of hydrops fetalis. Definitive diagnosis of alpha-thalassemia 1 carriers is difficult since there are few red cell abnormalities. Recently Chui et al. found that minute amounts of embryonic zeta-globin chains are present in adult hemoglobin of the Southeast Asian type of alpha-thalassemia 1 carriers. In this study, we screened 521 cord bloods for alpha-thalassemia 1. Hemoglobin analysis, including quantitation of Hb Bart's, was performed using the automated HPLC, alpha-thalassemia short program (VARIANT, Bio-Rad, Hercules, CA). Of these, 200 cord blood samples in which Hb Bart's was demonstrated were tested for the presence of zeta-globin chains by ELISA using labeled anti-zeta monoclonal antibody. Zeta-globin ranged between 0.21 and 0.83% in 19 specimens carrying alpha-thalassemia 1 gene. In the remaining 90 out of 109 specimens in which Hb Bart's was greater than 1.2%, zeta-globin was less than 0.17%. DNA analysis revealed the presence of normal alpha-genotype and other types of alpha-thalassemia including alpha-thalassemia 2 and Hb Constant Spring. One false positive was found in which the zeta-globin was 0.25% by ELISA but in which PCR indicated an alpha-thalassemia 2 heterozygote. Ninety-one samples with Hb Bart's of less than 1.2% by HPLC are most likely normal with a zeta-globin range between 0 and 0.14%. This study also showed that the frequency of alpha-thalassemia 1 in Bangkok is 3.65%.

Adult↗
PubMed1998

Renal function in adult beta-thalassemia/Hb E disease.

Beta-Thalassemia hemoglobin E (beta-thal/Hb E) is the commonest form of hemoglobinopathy in Thailand. Shortened red cell life span, rapid iron turnover and tissue deposition of excess iron are major factors responsible for functional and physiological abnormalities found in various forms of thalassemia. Increased deposition of iron had been found in renal parenchyma of thalassemic patients, but no systematic study of the effect of the deposits on renal functions has been available. The purpose of this study is to describe the functional abnormalities of the kidney in patients with beta-thal/Hb E and provide evidence that increased oxidative stress might be one of the factors responsible for the damage. Urine and serum samples from 95 patients with beta-thal/Hb E were studied comparing with 27 age-matched healthy controls. No difference in the creatinine clearance was observed. beta-thal/Hb E patients excreted significantly more urinary protein (0.8+/-0.5 vs. 0.3+/-0.1 g/day, p < 0.001). Aminoaciduria was found in 16 % of the patients. Analysis of urinary protein by SDS-PAGE electrophoresis and silver staining revealed abnormal pattern of protein with increased small molecular weight (<45 kD) bands. Morning urine analysis showed significant lower urine osmolality (578.3+/-164.6 vs. 762.4+/-169.9 mosm/kg, p < 0.001) in patients. Patients excreted more NAG (N-acetyl beta-D-glucosaminidase, 26.3+/-41.3 vs. 8.4+/-3.9 U/g Cr, p < 0.0001) and beta2-microglobulin, 124.3+/-167 vs. 71+/-65.5 microg/g Cr, p = 0.001. Plasma and urine MDA (malonyldialdehyde) levels were both raised (p < 0.0001). Nine patients were selected for renal acidification study. All were found to be normal, but showed poor response to DDAVP challenge (urine osmolality 533+/-71). This is the first report of renal tubular defects found associated with beta-thal/Hb E disease. The mechanism leading to the damage is not known but it might be related to increased oxidative stress secondary to tissue deposition of iron, as indicated by the raised levels of serum and urine MDA. It is not known whether these functional defects would have any long-term effects on the patients. Further studies are warranted and means of prevention of these defects should urgently be sought.

Acetylglucosaminidase↗
PubMed1998

Molecular basis of beta-thalassemia in the Maldives.

We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.

Algeria↗
PubMed1998

Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC.

The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT) system to study alpha-thalassemia and beta-thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of alpha-thalassemia and beta-thalassemia disorders, including Hb H, EA Bart's disease, and EF Bart's disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart's. The alpha-thalassemia short program was therefore developed and used to quantify Hb Bart's to detect alpha-thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia.

Adult↗
PubMed1997

The unusual pathobiology of hemoglobin constant spring red blood cells.

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other alpha-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV "normalizing" effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional alpha-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and alpha-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional alpha-thalassemic variants and found that in addition to oxidized beta-globin chains, oxidized alpha cs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized alpha cs- and beta-globin chains. The membrane alterations induced by alpha cs chains are more akin to those induced by beta A-globin chains than those induced by the alpha A-globin chains that accumulate in the beta-thalassemias. Thus, each globin chain, alpha cs, alpha A, beta A, appears to produce its own form of membrane perturbation.

Erythrocyte Membrane↗
PubMed1997

Presence of immunoglobulins, C3 and cytolytic C5b-9 complement components on the surface of erythrocytes from patients with beta-thalassaemia/HbE disease.

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employing a monoclonal antibody specific for a C5b-9 neoantigen. When C5b-9 positive cells from two patients were solubilized with detergent and subjected to sucrose density gradient centrifugation, the terminal complexes sedimented as 25-40S macromolecules, thus behaving as membrane C5b-9 complexes. The presence of C8 and C9 in these high molecular weight fractions was directly demonstrated by Western blotting. These results constitute the first demonstration that circulating diseased erythrocytes may carry low numbers of potentially cytolytic C5b-9 complement complexes which may be partly responsible for the known ionic disturbances found in thalassaemic cells. Both bound C3 and C5b-9 could promote removal of diseased cells in the reticuloendothelial system.

Adult↗
PubMed1997

An in vitro study on thalassemic erythroid precursors in liquid culture.

Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.

Cells, Cultured↗
PubMed1997

Alpha-thalassemia incidence in southern Thailand by restriction endonuclease analysis of globin DNA from placental blood at Songklanagarind Hospital.

The incidence of alpha-thalassemia has been studied previously based on the levels of Hb Barts' in cord blood. This method is an inadequate indicator of alpha-thalassemia. Thus in this study we use DNA analysis to get more accurate data. Hb Barts' was detected in placental blood samples from 15.5% of 375 infants born at Songklanagarind Hospital. The white blood cell DNA of 300 samples was studied for alpha-globin gene deletions by hybridization of DNA fragments digested by the restriction endonuclease Eco RI with specific 32P-labled zeta-globin gene probe. The incidence of alpha-thal 2 and alpha-thal 1 traits were 12.0% and 4.3%, with the gene frequencies 0.0650 and 0.0217 for -alpha/and --/, respectively. The incidence of HB CS trait was 5.8%, with the gene frequency of 0.0292 for alpha cs alpha/. We also found that the incidence of the triplicated zeta and triplicated alpha were 14.7 and 1.0%, with the gene frequencies of 0.0733 and 0.0050 for zeta zeta zeta/and alpha alpha alpha/, respectively. The DNA lesion of alpha-thalassemia in the south is similar to the study of Tanphaichitr et al (1988) in central Thailand. Knowledge of alpha-globin gene deletion would be useful for prenatal diagnosis of Bart's hydrops to prevent toxemia of pregnancy in the south of Thailand.

DNA↗
PubMed1997

Molecular and hematological characterization of HbE heterozygote with alpha-thalassemia determinant.

Hemoglobin E and alpha-thalassemia are prevalent in Thailand. The chance that an individual heterozygous for HbE also carries an alpha-thalassemia determinant is high. In this individual, the amount of HbE and other hematological parameters may be differed from that of usual observation. In this study, a total of 132 HbE heterozygotes were screened for alpha-thalassemia 1 gene deletion by the polymerase chain reaction. Out of 132 cases, 71 could be completely analyzed for hematologic parameters. Forty-three of 88 cases with HbE less than 25% as measured using microcolumn chromatography were positive for this gene deletion. In twenty of these 43 alpha-thalassemia 1 positive cases, the average values of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 10.6 g/ dl, 33.1%, 64.8 fl, 21.0 pg, 32.3 pg/dl, 18.6% and 17.4%, respectively. Eight of 9 alpha-thalassemia 1 negative cases were positive for alpha-thalassemia 2 gene deletion in Southern blot analysis. In this later group, hematological parameters were similar to that of the former. Co-inheritance of the Hb Constant Spring gene has no direct effect on the level of HbE. No alpha-thalassemia 1 gene was detected in the remaining 34 cases whose HbE were above 25%. The average amount of Hb, Hct, MCV, MCH, MCHC, RDW and HbE were 12.4 g/dl, 37.7%, 79.7 fl, 26.2 pg, 32.7 pg/dl, 25.8% and 28.5%, respectively. Therefore, screening for HbE level below 25% may be a convenient way of identifying parents of carrying alpha-thalassemia 1 determinant.

Erythrocytes↗
PubMed1997

Molecular and hematological characterization of Hb Tak and Hb Pyrgos in Thailand.

Two hemoglobin variants that migrate abnormally on gel electrophoresis were found in four unrelated Thai individuals. One variant that migrate faster than HbA but more slowly than Hb Bart's was detected in two heterozygotes. Another abnormal Hb migrating between HbA2 and HbF was found in one heterozygote and one compound heterozygote with HbE. In all cases, no microcytic anemia was observed. PCR amplification and direct DNA sequencing established that the first variant was caused by a missense mutation at codon 83 (GGC-GAC) that leads to Gly to Asp substitution previously described as the Hb Pyrgos in a Greek boy. The second variant was caused by an AC insertion at the termination codon that leads to synthesis of elongated beta-globin chain known as the Hb Tak. Beta globin gene haplotype analysis demonstrated that each variant was found on the same chromosome background in Thai individuals. The simple non-radioactive DNA assays based on allele specific polymerase chain reaction for the detection of these two Hb mutations in a routine laboratory are described.

Adult↗
PubMed1997

Beta-globin gene haplotypes in some minor ethnic groups in Thailand.

In order to provide population genetic data of various ethnic groups in Thailand, we have determined the type of hemoglobin by electrophoresis and the beta-globin gene haplotypes by PCR followed by restriction digestion in five small ethnic groups namely hill tribes, PhuTai, Chong, Lao Song and Sakai inhabiting in the north, northeast, east, central and south of Thailand, respectively. In each group, in addition to HbA and HbA2, the HbE, the most common hemoglobinopathy in Southeast Asia was detected at 2.5%, 51.6%, 84.0%, 8.6% and 11.8%, respectively. Haplotype analysis demonstrated that in all groups the beta A-globin gene was associated with various haplotypes and beta-globin gene frameworks. However, beta E -globin gene was associated with haplotypes ((-)+(-)+ + +(-)) and ((+)-(-)-(-)+(-)) on the beta-globin gene framework 2 in all ethnic groups except in Chong people whose the beta E-globin gene was mostly linked to haplotype ((-)+(-)++(-)+) and beta-globin gene framework 3 which was commonly found among Cambodian. It appears therefore that the Chong population is more related to Cambodian than Thai.

Ethnicity↗
PubMed1997

Detection of thalassemia genes using smeared blood film or leukocytes adhering to polysthylene fibers.

Presently genetic analyses for thalassemia types require relatively large amounts of heparinized blood (5 to 10 ml), and transport as well as degeneration of these sample is a problem in the developing world. We have developed a new method to simplify this procedure and obtain DNAs from small specimens. As experimental materials, thinly smeared blood on a glass slide or blood filtered with and adhered on polysthylene telephtalate (PST) fibers were used. These materials could be safely stored without interfering with DNA extraction for up to 3 months. The slide materials were digested with proteinase K, and DNA was extracted with Tris-EDTA-phenol:chloroform and precipitated with absolute ethanol. The PST specimens were washed with physiologic saline and treated in the same manner as described above. Products were easily amplified by PCR and digested with restriction endonucleases for beta thalassemia typing as well as for HLA-DQA1 gene typing. Results obtained by this method correlated well with previously reported incidences for thalassemia and HLA-DQA1 types in Thailand. This method can be used in the routine laboratory because it allows for stable and biosafe genetic analyses.

DNA↗