Labile iron pool as a parameter to monitor iron overload and oxidative stress status in ß-thalassemic erythrocytes.

BACKGROUND: Labile iron pool (LIP) is intracellular nonprotein bound iron that can generate oxygen radicals via the Fenton reaction resulting in oxidative cell damage. Therefore, quantitative measurement of LIP will be helpful for detecting and monitoring the toxic iron status in iron overloaded patients. This study demonstrated LIP level and its correlation to oxidative stress status in ß-thalassemic erythrocytes. METHODS: LIP and reactive oxygen species (ROS) level, numbers of erythrocyte vesicles and apoptosis were assayed by flow cytometric methods in 30 blood samples from ß-thalassemia/hemoglobin E patients and 17 blood samples from healthy volunteers with normal hemoglobin type. RESULTS: ß-thalassemic erythrocytes showed higher LIP level, defined as the difference in calcein fluorescent intensity of the cells treated with or without deferiprone, than normal erythrocytes (mean ± 2SD as 62.39 ± 39.58 versus 44.65 ± 35.86, P = 0.003). The LIP level above 67, a cutoff value of LIP level obtained from receiver operating characteristic curve analysis, had a significant positive correlation with oxidative stress status for ROS level (r = 0.90, P < 0.001) and also the amount of erythrocyte vesicles (r = 0.79, P = 0.002). In contrast, the LIP level showed a significant negative correlation with the patients' hemoglobin level (r = -0.66, P = 0.028). CONCLUSIONS: The LIP assay is suggested as an alternative test to monitor the magnitude of iron overload and its consequent oxidative stress in ß-thalassemia. LIP level may also be used as a marker for therapeutic response to iron chelation treatment. © 2018 International Clinical Cytometry Society.

Hematological parameters and red blood cell morphological abnormality of Glucose-6-Phosphate dehydrogenase deficiency co-inherited with thalassemia.

OBJECTIVE/BACKGROUND: Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and thalassemia are genetically independent hemolytic disorders. Co-inheritance of both disorders may affect red blood cell pathology to a greater extent than normally seen in either disorder alone. This study determines the prevalence and evaluates hematological changes of G-6-PD deficiency and thalassemia co-inheritance. METHODS: G-6-PD deficiency was screened from 200 male thalassemia blood samples using a fluorescent spot test. Hematological parameters and red blood cell morphology were evaluated among G-6-PD deficiency/thalassemia co-inheritance, G-6-PD deficiency alone, thalassemia alone, and normal individuals. RESULTS: G-6-PD deficiency was detected together with hemoglobin (Hb) E heterozygote, Hb E homozygote, ß-thalassemia trait, and ß-thalassemia/Hb E, α-thalassemia-2 trait, and Hb H disease. Hb level, hematocrit, mean cell volume, and mean cell Hb of G-6-PD deficiency co-inherited with asymptomatic thalassemia carriers show significantly lower mean values compared to carriers with only the same thalassemia genotypes. Higher mean red blood cell distribution width was observed in G-6-PD deficiency co-inherited with Hb E heterozygote, as with numbers of hemighost cells in G-6-PD deficiency/thalassemia co-inheritance compared to those with either disorder. Apart from Hb level, hematological parameters of co-inheritance disorders were not different from individuals with a single thalassemia disease. CONCLUSION: G-6-PD deficiency co-inherited with thalassemia in males was present in 10% of the participants, resulting in worsening of red blood cell pathology compared with inheritance of thalassemia alone.

Enhanced erythroid cell differentiation in hypoxic condition is in part contributed by miR-210.

Erythropoiesis, a process of erythroid production, is controlled by several factors including oxygen level. In this study, the effect of oxygen tension on erythropoiesis was investigated in K562 erythroleukemic cell line and erythroid progenitor cells derived from normal and ß-thalassemia/hemoglobin (Hb) E individuals. The enhanced erythroid differentiation specific markers including increased levels of α-, ß- and γ-globin gene expressions, numbers of HbF positive cells and the presence of glycophorin A surface marker were observed during cell culture under hypoxic atmosphere. The result also showed that miR-210, one of the hypoxia-induced miRNAs, was up-regulated in K562 and ß-thalassemia/HbE progenitor cells cultured under hypoxic condition. Inhibition of miR-210 expression leads to reduction of the globin gene expression and delayed maturation in K562 and erythroid progenitor cells. This indicated that miR-210 contributes to hypoxia-induced erythroid differentiation in both K562 cells and ß-thalassemia/HbE erythroid progenitor cells.

Rapid diagnosis of alpha-thalassemia by melting curve analysis.

alpha-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of alpha-globin protein. Couples who both carry the alpha-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the alpha-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for alpha-thalassemia diagnosis was developed by genotyping alpha-thalassemia-1, the Southeast Asian type (--(SEA)) and Thai type (--(THAI)) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the --(SEA) and --(THAI) deletions and two normal fragments, psizeta- and alpha2-globin gene. The primers were capable of distinguishing alpha-thalassemia 1 heterozygotes from alpha-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --(THAI), --(SEA), psizeta-globin, and alpha2-globin gene fragments were 79.9 +/- 0.2, 89.4 +/- 0.5, 92.8 +/- 0.2, and 85.0 +/- 0.2 degrees C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.

A smart model for clinical laboratory personnel development.

BACKGROUND: To become a quality clinical laboratory, personnel development is the most important factor. In order to achieve this goal, it should emphasize that clinical laboratory is not only a testing laboratory; it must be a knowledge-based service laboratory. A smart model for clinical laboratory personnel development under the Human Asset Development (HAD) program had been launched since 2003. OBJECTIVE: To strengthen the competency of clinical laboratory personnel, an appropriate model was developed and apply to the clinical laboratory personnel. MATERIAL AND METHOD: Medical technologist who currently worked in clinical laboratory participated in this study. The proposed model consisted of 3 phases. 1) The knowledge providing via update and refresher courses. 2) Application of learned knowledge to practice under close supervision. 3) Training on special topic and self oriented research activity. RESULTS: The outcome of 5 years project was evaluated. After the first phase, they were able to identify and solve their own troublesome under ours close supervision. There were 25 projects presented within 3 years. The last phase, they were very constructive. Nine projects of self created had been presented. Those projects contained clear objectives and were able to implement. CONCLUSION: The smart model for clinical laboratory personnel development leaded to many self created projects in a few years. Thus, this implies its important role in human resource development that should be continued. The keys index of success were ours strong intention, with providing motivation and periodically encouragement to the participants, and keep going on consistently.

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice.

BACKGROUND: beta-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate beta-globin chain production. The excessive unbound alpha-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis. DESIGN AND METHODS: In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival. RESULTS: The alpha/beta-globin chain ratio in beta(IVSII-654)-thalassemic mice, 1.26+/-0.03, was significantly higher than that of wild type mice, 0.96+/-0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human beta(E)-globin transgene, with a more balanced globin chain synthesis, 0.92+/-0.05. Moreover, transgenic mice harboring 8 extra copies of the human beta(E)-globin transgene showed inversely imbalanced alpha/beta-globin synthesis ratio, 0.83+/-0.01, that resulted in a mild beta-thalassemia phenotype due to the excessive beta-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of beta(IVSII-654)-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice. CONCLUSIONS: Imbalanced alpha/beta-globin chain as a consequence of either reduction or enhancement of beta-globin chain synthesis can cause abnormal red blood cell properties in mouse models.

Detection and haplotype differentiation of Southeast Asian alpha-thalassemia using polymerase chain reaction and a piezoelectric biosensor immobilized with a single oligonucleotide probe.

DNA-based diagnosis of alpha-thalassemias routinely relies on polymerase chain reaction (PCR) and gel electrophoresis. Here, we developed a new procedure for the detection and haplotype differentiation of Southeast Asian (SEA) alpha-thalassemia using a 3-primer system for PCR coupling with a DNA-based piezoelectric biosensor. PCR products amplified from genomic DNA were differentiated directly by using a quartz crystal microbalance immobilized with a single oligonucleotide probe. The frequency changes after hybridization of the PCR products amplified from a representative sample of normal alpha-globin, SEA alpha-thalassemia heterozygote, and homozygote were 206+/-11, 256+/-5, and 307+/-3 Hz, respectively. The fabricated biosensor was evaluated through an examination of 18 blind specimens. It could accurately discriminate between normal and SEA alpha-thalassemic samples, which suggests that this biosensor system is a promising alternative technique to detect SEA alpha-thalassemia because of its specificity and less hazardous exposure as compared with conventional methods.

Inhibition of alpha-globin gene expression by RNAi.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.

Analysis of breast cancer susceptibility genes BRCA1 and BRCA2 in Thai familial and isolated early-onset breast and ovarian cancer.

Here we report the study on BRCA1 and BRCA2 mutations in 12 Thai breast and/or ovarian cancer families and 6 early-onset breast or breast/ovarian cancer cases without a family history of cancer. Five distinct rare alterations were identified in each gene: four introducing premature stop codons, one in-frame deletion, two missense changes, two intronic alterations and one silent rare variant. The BRCA1 or BRCA2 truncating mutations were detected in four of seven patients with familial or personal history of breast and ovarian cancer, in one of four isolated early onset breast cancer cases and in none of seven breast cancer site specific families. The BRCA1 and BRCA2 mutation yield in Thai patients is consistent with that reported from Europe and North America in similar groups of patients, being particularly high in individuals with personal or family history of breast and ovarian cancer. The BRCA1 and BRCA2 alterations found in this series are different from those identified in other Asian studies, and all but two have never been reported before. We report at least three novel deleterious mutations, the BRCA1 3300delA, BRCA1 744ins20 and BRCA2 6382delT. One in-frame deletion was also found, the BRCA2 5527del9, which seggregated within family members of breast-only cancer patients and was thought to be a cancer-related mutation. BRCA1 3300delA and Asp67Glu alterations were detected each in at least two families and thus could represent founder mutations in Thais.

Membrane heme as a host factor in reducing effectiveness of dihydroartemisinin.

Plasmodium falciparum infecting alpha-thalassemic erythrocytes are resistant to artemisinin and its derivatives. Binding of the drug to hemoglobin H resulting in drug inactivation was previously demonstrated. We now show that an additional host factor, membrane heme, significantly accounted for decreased antimalarial activity of artemisinin. The antimalarial activity of dihydroartemisinin in the presence of normal and thalassemic erythrocyte membranes showed a correlation with the heme content of the membrane (r(2)=0.466, P<0.01). The correlation was more clearly seen when the drug effectiveness was correlated with the heme content of alpha-thalassemic membrane (r(2)=0.636, P<0.01). However, the drug effectiveness showed no correlation to ferrozine-reactive (free or non-heme) iron content (r(2)=0.0001, P>0.05). alpha-Thalassemic erythrocytes contained higher amounts of membrane heme (11.04+/-8.96 nmol/mg membrane protein) than those from normal and beta-thalassemia/HbE erythrocytes (2.68+/-1.28 and 3.98+/-3.98 nmol/mg membrane protein, respectively, P<0.01). Loss of drug effectiveness was also correlated with increment of heme content in membrane prepared from normal erythrocytes treated with phenylhydrazine. It is concluded that heme in both normal and thalassemic erythrocyte membranes is an important factor in drug inactivation.