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Pornprasert S.,Chiang Mai University | Sukunthamala K.,Health Promoting Hospital Chiang Mai | Kunyanone N.,Chiang Rai Hospital | Sittiprasert S.,Chiang Rai Hospital | And 6 more authors.
Journal of the Medical Association of Thailand | Year: 2010

Background: Noninvasive prenatal diagnosis based on detection of fetal cell-free DNA is hampered when mother and father are both carriers for the same autosomal recessive mutation. Objective: To compare the diagnosis of Bart's hydrops fetalis using conventional Gap-PCR analysis of fetal cells/tissues with the measurement of quantitative difference (ΔCT) between α-thalassemia-1 SEA type deletion gene (CT-mutant) and wild type α-globin gene (CT-wild type) in plasma of pregnancies by using the Taqman real-time quantitative PCR. Material and Method: Plasma DNA samples were collected from three groups of pregnancies whose fetuses have known thalasemia status (7 normal, 11 heterozygote α-thalassemia-1 SEA type deletion, and 7 Bart's hydrops fetalis). The α-thalassemia-1 SEA type deletion gene and wild type α-globin gene were quantified by using Taqman real-time quantitative PCR and then the ΔCT was analyzed by subtracting the CT-mutant from CT-wild type. Results: Mean ΔCT values were not significantly different among the three groups. However, women whose fetuses were diagnosed as Bart's hydrops fetalis had a higher proportion (43%) of plasma DNA samples that had negative ΔCT value than women whose fetuses were diagnosed as normal or heterozygote α-thalassemia-1 SEA type deletion (0 and 27%, respectively). Conclusion: Further investigations are needed to improve the diagnosis of Bart's hydrops fetalis using fetal cell-free DNA.


Pornprasert S.,Chiang Mai University | Panyasai S.,University of Phayao | Waneesorn J.,Regional Medical science Center | Kongthai K.,Health Promoting Hospital Chiang Mai | Singboottra P.,Chiang Mai University
Clinical Laboratory | Year: 2012

Background: Gel-electrophoresis and ethidium bromide are not ideally suited to large scale analysis in clinical laboratories. Methods: Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) specific for Hb CS was performed in 10 blood samples from normal individuals and 61 samples containing a peak of Hb CS when analyzed by capillary electrophoresis. Heterozygosity of Hb CS was identified using SYTO9 and high resolution melting (HRM) analysis method. Results: Specific peak heights of amplified fragments of wild type and Hb CS alleles were observed in the heterozygote. Only one peak height of amplified fragments of the wild type allele was observed in the normal individual while only one peak height of amplified fragments of Hb CS allele was observed in the homozygote. HRM analysis interpretation results were completely consistent with the interpretation results from gel-electrophoresis. Conclusions: SYTO9 HRM analysis may be used as an alternative for rapid diagnosis of heterozygosity of Hb CS.


Pornprasert S.,Chiang Mai University | Panyasai S.,University of Phayao | Kongthai K.,Health Promoting Hospital Chiang Mai | Treesuwan K.,Chiang Mai University
Hemoglobin | Year: 2012

Hb S [β6(A3)Glu→Val, GAG>GTG] is a β-globin gene variant that has a very low incidence in the Thai population. Coinheritance of Hb S and β0-thalassemia (β-thal) can result in severe clinical conditions. This study reports the case of a Thai patient with a compound heterozygosity for Hb S and β0-thal codon 17 (A>T). His hemoglobin (Hb), hematocrit (packed cell volume, PCV), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) levels were all less than the lower limits, while red cell distribution width (RDW) was higher than the upper limit. Levels of Hbs S, F and A2 detected by high performance liquid chromatography (HPLC) were comparable to those from capillary electrophoresis (CE). As Hb S has a similar electrophoretic mobility and the HPLC profile is also similar to those of Hb Tak [β147, Term→Thr (AC)] and Hb D-Punjab [β121(GH4) Glu→Gln, GAA>CAA], DNA sequencing was then performed. This was to detect β0-thal, and to differentiate Hb S from the Hb Tak and Hb D-Punjab mutations. The β0-thal codon 17 and Hb S mutations were detected indicating that coinheritance of these two mutations can be found in the Thai population. Therefore, to provide proper clinical management and genetic counseling of this rare case, DNA analysis should be performed in all cases when a peak at the S-window is detected by HPLC or CE. Copyright © Informa Healthcare USA, Inc.


Pornprasert S.,Chiang Mai University | Panyasai S.,University of Phayao | Kongthai K.,Health Promoting Hospital Chiang Mai
Clinical Chemistry and Laboratory Medicine | Year: 2012

Background: Hemoglobin (Hb) A2 is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA2 levels and capillary electrophoregrams for associa-tions of heterozygote of Hb Hope with α-thalassemia nor β-thalassemia. Methods: Levels of HbA0, HbA2 and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/a-thalassemia-1 SEA type deletion and 2 Hb Hope/β0- thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups. Results: Artifactually elevated HbA 2 levels (≥4%) were found in both groups of heterozygous Hb Hope and Hb Hope/α-thalassemia-1 SEA type deletion. Manual corrections were performed by adjusting baselines, and results showed that means of HbA 2 in both groups decreased from 4.47 % and 4.03 % to 1.93 % and 1.77 %, respectively. The highest levels of HbA2 and Hb Hope were observed in samples with Hb Hope/α0-thalassemia. Moreover, HbA 0 was not observe in these cases. Conclusions: The elevation of HbA2 in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect β-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA0, HbA2 and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/βdeg;-thalassemia. © 2012 by Walter de Gruyter. Berlin. Boston.


Pornprasert S.,Chiang Mai University | Panyasai S.,University of Phayao | Waneesorn J.,Regional Medical science Center 10 Chiang Mai | Kongthai K.,Health Promoting Hospital Chiang Mai | Singboottra P.,Chiang Mai University
International Journal of Laboratory Hematology | Year: 2012

Introduction: Capillary electrophoresis (CE) is a high-resolution method for detection of hemoglobin Constant Spring (Hb CS). Methods: The levels of Hb CS quantified by CE were compared among three groups of samples including heterozygote and homozygote of Hb CS as well as Hb H-CS disease classified by DNA molecular diagnosis. The mean corpuscular volume (MCV) of red blood cells was also analyzed among these three groups. Results: Mean±SD of Hb CS level of the homozygote was not significantly different from that of the Hb H-CS disease (1.9±1.8 vs. 2.8±1.3, P=0.13), but it was significantly higher than that of the heterozygote (1.9±1.8 vs. 0.4±0.2, P=0.007). The MCV <70fL was found in Hb H-CS disease only. Conclusion: CE is the preferable method for screening of heterozygote and homozygote of Hb CS. Moreover, in conjunction with a lower MCV (<70fL), this approach provided a high resolution to identify Hb H-CS disease. © 2011 Blackwell Publishing Ltd.


Srithep S.,Chiang Mai University | Kongthai K.,Health Promoting Hospital Chiang Mai | Pornprasert S.,Chiang Mai University
Clinical Laboratory | Year: 2013

Background: The combination of an x-linked hematologic disorder with a heterozygous α-thalassemia-1 Southeast Asian (SEA) type deletion might lead to severe anemia in male infants. This study is to develop a simple and cost-effective single tube multiplex real-time PCR for the diagnosis of α-thalassemia-1 SEA type deletion and detect fetal gender. Methods: Multiplex real-time polymerase chain reaction (PCR) for the detection of α-thalassemia-1 SEA type deletion gene, wild type α-globin gene, and sex-determining region Y (SRY) gene was validated by analysis of 95 cord blood samples (60 normal individuals, 28 heterozygous of α-thalassemia-1 SEA type deletion and 7 Bart's hydrops fetalis). The change in threshold cycle (ΔCT) was analyzed by subtracting the CT-mutant from CT-wild type. Results: Mean ΔCT values were significantly different among these three groups, and a SRY gene determination was 100% in concordance with fetal genders. Furthermore, analysis of fetal gender did not affect the ΔCT of α-thalassemia-1 SEA detection. Conclusions: Combined α-thalassemia-1 SEA type detection and fetal gender determination in a single-tube multiplex real-time PCR is an alternative assay for a conventional method for the diagnosis of α-thalassemia-1 SEA deletion and fetal gender.


Pornprasert S.,Chiang Mai University | Treesuwan K.,Chiang Mai University | Punyamung M.,Chiang Mai University | Kongthai K.,Health Promoting Hospital Chiang Mai
Hemoglobin | Year: 2012

The α-thalassemia-1 (α-thal-1) Southeast Asian ( SEA) type deletion, β-thalassemia (β-thal) and Hb E [β26(B8)Glu→Lys, GAG>AAG] are the most common genetic disorders in Southeast Asian populations. Mean corpuscular volume (MCV) <80.0 fL with normal hemoglobin (Hb) is used for screening α- and β-thal, and a Hb E level of less than 25.0 is used for predicting α-thal-1 in Hb E trait. Thus, levels of Hb, MCV and Hb A2/E were reviewed and compared between the SEA type deletion co-inherited with β-thal trait (n 61), with Hb E trait (n 102) or homozygous Hb E (n 13) and β-thal trait (n 636), Hb E trait (n 544) or homozygous Hb E (n 83), respectively. When comparing the values of all three analyzed hematological parameters, only the SEA/βE values were shown to be significantly lower than those of Hb E trait. Furthermore, at a cut-off value of Hb A2/E of 21.54, 95.0 of the SEA/βE had Hb A2/E levels lower than this cut-off value, while 94.0 of Hb E trait had Hb A2/E at higher levels. Accordingly, the Hb A2/E level at 21.54 is the best indicator for predicting co-inheritance of the α-thal-1 SEA/ deletion and Hb E trait. © 2012 Informa Healthcare USA, Inc.


Pornprasert S.,Chiang Mai University | Sukunthamala K.,Health Promoting Hospital Chiang Mai
European Journal of Haematology | Year: 2010

The Β0-thalassemia/Hb-E causes a wide range of severe conditions. A high medical cost is incurred in severe cases. Thus, the prevention of new cases of Β0-thalassemia/Hb-E is required. The aim of this study is to use the SYTO9 and SYBR GREEN1 high-resolution melting (HRM) analysis for prenatal diagnosis of Β0-thalassemia/Hb-E. DNA samples were extracted from amniotic fluid or cord blood of 11 pregnancies whose fetuses were at risk for Β-thalassemia/Hb-E. PCR products from multiplex amplification refractory mutation system PCR for the detection of β0-thalassemia mutations at codons 17(A>T), 41/42(-TCTT), and 71/72(+A) and from amplification refractory mutation system PCR for the detection of Hb-E were characterized by SYTO9 HRM analysis. Moreover, β0-thalassemia 3.5- kb deletion was detected using real-time PCR with SYBR GREEN1 HRM analysis. Seven of 11 fetuses (64%) were diagnosed as β0-thalassemia/Hb-E (4 fetuses with mutation at codon 17, 2 with mutation at codon 41/42, and 1 with 3.5- kb deletion). Results from HRM analysis were completely consistent with those from fetal blood samplings analyzed at the time of delivery or pregnancy termination using HPLC. Therefore, the HRM analysis is easy to use. It is simple, flexible, non-destructive and has superb sensitivity and specificity. This approach might facilitate the laboratory diagnosis and genetic counseling for regions with a high prevalence ofβ0-thalassemia/Hb-E. © 2010 John Wiley & Sons A/S.


Pornprasert S.,Chiang Mai University | Kasemrad C.,Chiang Mai University | Sukunthamala K.,Health Promoting Hospital Chiang Mai
Hemoglobin | Year: 2010

High performance liquid chromatography (HPLC) on fresh lysates is the standard test for identification of thalassemia. Samples in the form of dried blood spot(s) (DBS) mailed to reference laboratories where HPLC is available could be an alternative. Hemoglobin (Hb) on DBS at day 1, 7, 15 and 30 were analyzed by HPLC and compared to those analyzed from fresh liquid whole blood at day 0. A 100 consistent interpretation of β-thalassemia (β-thal) trait and β-thalHb E disease between liquid whole blood and DBS was observed when analyzing Hb A2 on DBS at a level of 2.79.9 in conjunction with a lower MCV (<80 fL) and MCH (<27 pg) and analyzing β-thalHb E by using Hb E and Hb F at a level of 3048 and >10, respectively. Therefore, our results show that detection of thalassemia carriers using DBS is possible and is the alternative of choice in a low resource setting. © 2010 Informa UK Ltd.


Wongnoi R.,Chiang Mai University | Oberdorfer P.,Chiang Mai University | Sirivatanapa P.,Chiang Mai University | Phanpong C.,Health Promoting Hospital Chiang Mai | Pornprasert S.,Chiang Mai University
Current HIV Research | Year: 2013

The effects of antiretroviral (ARV) drugs administered to HIV-infected pregnancy on hematological parameters and hemoglobin (Hb) synthesis in ARV-exposed newborns with and without thalassemia carrier and of ARV drugs in worsening anemia in thalassemia carrier newborns are not well understood. Cord blood samples were collected from newborns of HIV-infected and -uninfected pregnancies. Hematological parameters and hemoglobin typing were analyzed by automated blood counter and capillary electrophoresis (CE), respectively. In the group of thalassemia carrier, the ARV-exposed newborns had significantly lower mean levels of red blood cell counts and hematocrit and had significantly higher mean levels of MCH than the ARV-unexposed newborns. Similar results were found in the group of newborns without thalassemia carrier. There were no statistical differences in mean levels of Hb-A2, Hb-A, Hb-F and Hb-E (when applicable) in ARV-exposed and -unexposed newborns either with or without thalassemia carrier. However, ARVexposed newborns who were thalassemia carriers had the lowest levels of hemoglobin and hematocrit when compared to the other groups. Therefore, ARV drugs used for prevention of HIV-mother-to-child transmission (HIV-MTCT) altered hematological parameters but did not affect hemoglobin synthesis in newborns with and without thalassemia carrier. However, thalassemia and ARV drugs might have synergetic effect in inducing severe anemia. © 2013 Bentham Science Publishers.

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