Thawalwong Ratanasiri

A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting.

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.

Prevention of severe thalassemia in northeast Thailand: 16 years of experience at a single university center.

To demonstrate the performance of thalassemia prevention in northeast Thailand during 1993–2008.

Simultaneous PCR detection of β – thalassemia and α – thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome

Abstract Objective: To establish a rapid PCR method for simultaneous detection of β-thalassemia and α-thalassemia 1 genes for diagnosis of complex αβ-thalassemia syndrome. Design and methods: Using multiplex allele specific PCR approach, we evaluated a simultaneous detection of the SEA type α-thalassemia 1 and the common Southeast Asian β-thalassemia and hemoglobin E genes. The system was tested on known cases of double heterozygote for α- and β-thalassemias and in a prenatal diagnosis of complex αβ-thalassemia syndrome. Results: Co-inheritance of α-thalassemia 1 (SEA type) with each of the common β-thalassemia genes in Southeast Asian and with hemoglobin E could be identified in a single PCR reaction. A successful application of this simultaneous detection system in prenatal diagnosis of a complex thalassemia syndrome caused by an EFBart’s disease was demonstrated in a Thai family. Conclusion: We have shown that correct diagnosis of double heterozygosity for α-thalassemia 1 and β-thalassemia or hemoglobin E could be obtained using a simultaneous multiplex PCR. These rapid PCR assays would facilitate characterization and prenatal diagnosis of complex thalassemia syndromes in the regions where both α- and β-thalassemias and hemoglobin E are common.

Non-invasive fetal sex determination using a conventional nested PCR analysis of fetal DNA in maternal plasma.

BACKGROUND In order to provide a reliable non-invasive method for fetal sex determination in a routine setting, we evaluated the possibility of identifying the fetal Y chromosome-specific sequence in maternal plasma using conventional PCR analysis. METHODS Fetal gender was determined in 31 pregnant women including one with a dizygotic twin pregnancy between 7 and 32 weeks of gestation using DNA extracted from 200 microl of each maternal plasma. The 198 bp SRY gene-specific sequence on Y chromosome and the 261 bp ATL1 gene-specific sequence on X chromosome were co-amplified in a multiplex nested PCR manner. The result was confirmed by routine analysis of fetal tissue obtained by invasive procedure or examination of newborns after delivery. RESULTS The 198 bp SRY-specific sequence was detected in 15 plasma samples obtained from pregnant women carrying male fetuses. In the remaining cases bearing female fetuses, only the 261 bp ATL1 gene sequence was detected, producing 100% sensitivity and specificity of fetal sex prediction. The result was completely concordant with the conventional fetal tissue analysis and examination of the newborns after delivery. CONCLUSIONS A conventional nested PCR analysis of maternal plasma could be used for accurate fetal gender detection and enable a reliable prospective non-invasive fetal sex determination which should enhance prenatal diagnostic options especially for sex-linked disorders.

Analysis of fetal blood using capillary electrophoresis system: a simple method for prenatal diagnosis of severe thalassemia diseases

Introduction:  Prenatal diagnosis of severe α‐ and β‐thalasssemia diseases is usually performed by DNA analysis.

Development and Application of a Real‐Time Quantitative PCR for Prenatal Detection of Fetal α0‐Thalassemia from Maternal Plasma

Abstract:  In order to provide a noninvasive prenatal diagnosis of α0‐Thalassemia (Southeast Asian [SEA] deletion), we have developed a real‐time quantitative semi‐nested polymerase chain reaction (PCR) method for identifying the fetal α0‐Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 μL plasma from 13 pregnant women during 8–20 weeks of gestation who carried fetuses with normal (2), α0‐Thalassemia carrier (8), Hb H disease (1), and homozygous α0‐Thalassemia (Hb Barts hydrops fetalis (2). The α0‐Thalassemia was detected using a two‐step PCR. Plasma DNA was amplified conventionally using α0‐Thalassemia‐specific primers and a portion of the first PCR product was subjected to a semi‐nested real‐time q‐PCR using the SYBR green I chemistry for fluorescence detection. Calibration curve for α0‐Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an α0‐Thalassemia carrier. Differences in the CT (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, α0‐Thalassemia carrier, Hb H disease, and homozygous α0‐Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of α0‐Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.

Complex interaction of Hb Hekinan [alpha27(B8) Glu-Asp] and Hb E [beta26(B8) Glu-Lys] with a deletional alpha-thalassemia 1 in a Thai family.

Abstract: Hemoglobin (Hb) Hekinan (α27; Glu–Asp) is a rare α‐chain variant found mainly in Japanese and Chinese whereas Hb E (β26; Glu–Lys) is common among Southeast Asians. We report a hitherto undescribed condition in which these two variants co‐segregate. The proband was a 25‐yr‐old Thai woman who was encountered with the presence of mild hypochromic microcytosis with Hb 8.2 g/dL, hematocrit (Hct) 26.0%, Mean Corpuscular Value (MCV) 68.6 fL, Mean Corpuscular Hemoglobin (MCH) 21.6 pg and Mean Corpuscular Hemoglobin Concentration (MCHC) 31.5 g/dL. Although Hb electrophoresis at alkaline pH did not show any abnormal band except Hb E in addition to Hb A, high performance liquid chromatography analysis revealed abnormal peaks at the Hb A and Hb E positions. DNA analysis of the proband revealed a GAG–GAT mutation at codon 27 of the minor α1‐globin gene for Hb Hekinan in trans to the South‐east Asian (SEA) deletional α‐thalassemia 1 determinant and a GAG–AAG mutation at codon 26 of the β‐globin gene for Hb E. She was therefore a triple heterozygote for these three anomalies. Family study identified that her mother was a double heterozygote for Hb Hekinan and Hb E without α‐thalassemia whereas her father was a classical Hb H disease patient. The genotype–phenotype relationship observed in this Thai family with complex hemoglobinopathies is presented and a simple DNA assay based on the polymerase chain reaction methodology for rapid diagnosis of Hb Hekinan is described.

Variability of Nuchal Translucency Measurement

Objectives: To evaluate intra- and inter-observer variations of nuchal translucency (NT) measurements and study the duration of measurements. Subjects: One hundred and forty-seven singleton pregnant women with 10–14 weeks of gestation who had attended antenatal clinic during January 1st, 2000–August 31st, 2001 were included. Methods: Crown-rump length and NT were measured three times for each woman. Then, another examiner who was unaware of the previous results did the other three measurements. The duration of measurements was recorded. Intra-observer and inter-observer variations were analyzed by repeated ANOVA and paired t test, respectively. The correlation of NT measurements within each observer and between paired observers was evaluated by intraclass correlation coefficients (ICC). Kappa statistic was calculated for agreement. Results: Intra-observer variation of all 10 examiners at three institutes was varied from 0.20 ± 0.27 to 0.33 ± 0.41 mm. Inter-observer variation at Chiang Mai University was 0.40 ± 0.37 mm which was the highest value compared with those at Prince of Songkla and Khon Kaen University (p < 0.05). The mean duration of measurements was in range of 8–12 min. The intra-observer repeatability of the first two measurements showed ICCs varying from 0.61 to 0.94. The inter-observer repeatability of paired examiners showed a variation in ICC from 0.28–0.90. The kappa value expressing the intra- and inter-observer repeatability as being >95th or ≤95th percentile was 0.73 and 0.72, respectively. Conclusions: NT measurement was reproducible for small variation and good agreement. The duration of measurements was acceptable.

Postterm pregnancy: computer analysis of the antepartum fetal heart rate patterns

The purpose of this study was to establish reference ranges for numerical fetal heart rate (FHR) data in postterm pregnancy and to compare them with the patterns of fetuses under undisturbed condition at term. FHR was analysed on‐line by Sonicaid Computer System 8000. A statistically significant decrease in the number of accelerations and decrease of variation in postterm pregnancy was observed. The duration of high variation (high episodes) in the 42nd week of gestation was statistically lower than in the pregnancy at term. These observations should be taken into account by clinicians in the interpretation of FHR records in postterm pregnancy.

Accurate prenatal diagnosis of Hb Bart's hydrops fetalis in daily practice with a double-check PCR system.

Hemoglobin (Hb) Bart’s hydrops fetalis is a fatal condition associated with homozygous α⁰-thalassemia. Prenatal diagnosis of the disease is usually done by gap-PCR; however, misdiagnosis can occur with allelic dropout. Diagnosis using more than one method is preferred. We describe a double-check PCR assay for accurate prenatal diagnosis. The study was conducted on 64 fetuses at risk of homozygous α⁰-thalassemia encountered at our routine thalassemia diagnosis laboratory. Chorionic villus sample (CVS), amniotic fluid or fetal blood specimens were obtained from pregnant women at risk and analyzed by two PCR methods. In the first method, the SEA α⁰-thalassemia deletion of parents and fetuses were determined by gap-PCR routinely run in our laboratory. In another method, two specific fragments located 5′ to the ζ2 gene (XbaI fragment) and the α2-globin gene (RsaI fragment) together with the gap-PCR fragment were multiply co-amplified to determine the presence or absence of normal and α⁰-thalassemia alleles. The molecular diagnosis of α⁰-thalassemia was possible in all 64 fetuses using the two PCR approaches. The final diagnoses included 13 normal, 29 unaffected heterozygote and 22 homozygote α⁰-thalassemia fetuses.The two PCR assays disclosed no discordant result in the diagnosis of the Hb Bart’s hydrops fetalis caused by α⁰-thalassemia.The combined PCR assay for gap-PCR, ζ2 XbaI and α2 RsaI fragments, described here, is simple, accurate and applicable in the prenatal diagnosis of Hb Bart’s hydrops fetalis in a routine setting.