Screening For Α-thalassemia Research Articles

Microcolumn and coelution hydration of oil seal blood spot for efficient screening of newborn α-thalassemia via chip isoelectric focusing

BackgroundThe global prevalence of α-thalassemia necessitates effective newborn screening strategies due to its severe clinical consequences. Traditional methods such as liquid chromatography (LC), capillary electrophoresis (CE), and isoelectric focusing (IEF) face limitations, including low separation efficiency, poor sensitivity for detecting Hb Bart’s, and time-intensive operations, particularly with dried blood spots (DBS). These limitations hinder timely and accurate screening. This study addresses the need for a more efficient, sensitive, and rapid method for detecting Hb Bart’s in newborns. ResultsWe enhanced IEF separation and sensitivity by designing a microfluidic IEF (mIEF) system with shortened columns and employing a coelution sample loading technique using oil-sealed blood spots for rapid sample pretreatment. Our experiments demonstrated significant improvements: the total analysis time was reduced from 1110 minutes (IPG IEF) and 46 minutes (LC) per batch to 36 minutes per batch. For individual samples, the focusing time decreased from 6 minutes (previous mIEF) to 3 minutes, with the microcolumn length shortened by 50%, from 30 mm to 15 mm. The developed method showed excellent consistency with clinical Bart’s detection and PCR diagnosis, achieving 100% sensitivity and 98% specificity for α-thalassemia screening. Significance and NoveltyOur novel mIEF method provides an efficient, sensitive, and rapid tool for screening newborns for α-thalassemia. This advancement addresses the limitations of traditional techniques, improving early diagnosis and intervention strategies and ultimately enhancing health outcomes for at-risk newborns.

Prenatal diagnostic errors in hemoglobin Bart's hydrops fetalis caused by rare genetic interactions of α-thalassemia.

To describe rare genetic interactions of α-thalassemia alleles causing Hb H disease and Hb Bart's hydrops fetalis which could lead to diagnostic errors in a routine practice. Hematological and molecular characterization were carried out in a Thai family with a risk of having fetus with Hb Bart's hydrops fetalis. Both parents were found to be the thalassemia intermedia patients associated with unusual forms of Hb H disease. DNA analysis of common α-thalassemia mutations in Thailand identified α+-thalassemia (-α3.7 kb del) and unknown α0-thalassemia in the father and α0-thalassemia (--SEA) with unknown α+-thalassemia in the mother. Fetal DNA analysis unlikely identified a homozygosity for α0-thalassemia (--SEA/--SEA). Further analysis identified that the father carried a rare South African α0-thalassemia in combination with α+-thalassemia (--SA/-α), whereas the mother was a patient with Hb H-Queens Park disease (--SEA/ααQP). The fetus was, in fact, a compound heterozygote for (--SA/--SEA). As shown in this study, routine screening for α-thalassemia at prenatal diagnosis in the region should include both common and rare α0-thalassemia alleles found in the population to effectively prevent a fatal condition of Hb Bart's hydrops fetalis syndrome.

Αlpha-thalassemia genotypes in Vietnam: a report of 12,030 pregnant women and their husbands performing prenatal screening for alpha-thalassemia

BACKGROUND Αlpha (α)-thalassemia is a global health concern, and improving screening methods is crucial for disease prevention. This study aimed to assess α-thalassemia genotypes and evaluate the effectiveness of various thresholds for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in prenatal screening for α-thalassemia.
 METHODS This cross-sectional study included pregnant women and their husbands who underwent prenatal screening for thalassemia at the National Hospital of Obstetrics and Gynecology, Vietnam from January 2012 to August 2021. Blood samples were collected and analyzed using the strip assay technique, which can detect 21 common mutations in the α-globin gene and 22 common mutations in the beta-globin gene.
 RESULTS Of the 12,030 participants, 931 were identified as having α-thalassemia, with --SEA, -α3.7, and -α4.2 being the most common mutations. When examining different thresholds of MCV and MCH, MCV <85 fL and MCH <28 pg had a lower missing rate than MCV <80 fL and MCH <27 pg, respectively. MCH <28 pg showed the highest sensitivity in screening for α-thalassemia. MCV <85 fL showed the lowest positive predictive value (PPV). The combination of MCV <80 fL and MCH <27 pg showed the lowest sensitivity in screening for α-thalassemia but the highest PPV among all thresholds.
 CONCLUSIONS Optimizing the screening methods for α-thalassemia is important for preventing and managing the disease in the community. These findings have important implications for thalassemia prevention and management programs and may contribute to reducing the burden of thalassemia in the global population.

Impact of age-dependent red blood cell parameters on α-globin gene genotyping in children.

When screening for α-thalassemia in children, adult cut-offs for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are generally applied to guide genetic evaluation. However, the normal ranges for MCV and MCH are lower in children than in adults, so we hypothesized that using age-matched cut-offs could lead to a more rational diagnostic strategy. The aim of this study was to evaluate if age-matched cut-offs could be applied advantageously. Data on children referred to a hemoglobin fractionation at the Department of Clinical Biochemistry, Aarhus University Hospital between 2016-2021 were identified in the laboratory information system. α-globin gene (HBA1/HBA2) genotyping was performed using multiplex gap-polymerase chain reaction. A total of 387 children were identified. HBA1/HBA2-genotyping was performed in 207 children (53%), and α-thalassemia was diagnosed in 47 children (23%) with -α3.7/αα being the predominant genotype (13%). We found that 23 children had MCV and MCH levels in the normal age-matched range, and two of these children (9%) were α+ thalassemia carriers with three functional α-globin genes. Using age-specific cut-off levels resulted in a reduction of 23 (11%) genotypes performed. In conclusion, applying age-matched cut-offs for MCV and MCH when screening children for α-thalassemia lead to 11% fewer genotypes performed while 9% carriers of α+ thalassemia (of the medically innocuous genotype -α3.7/αα) would have been overlooked.

Establishment of pregnant-specific intervals for hemoglobin (Hb) A2, HbF and cut-off points for HbA2 for thalassemia in Chongqing, China.

To analyze pregnant-specific intervals for hemoglobin A2 (HbA2), hemoglobin fetal (HbF), and cut-off points of HbA2 for thalassemia in Chongqing, China. Between September 2015 and April 2019, the study recruited 10039 individuals of reproductive age. Of which, 4399 healthy normal individuals were selected to determine reference values for HbA2 and HbF. The remaining 5640 individuals suspected of thalassemia were included to explore the cut-off points of HbA2 for thalassemia. The reference values of HbA2 in males was 2.3-3.2%, in females was 2.1-3.1%, and in pregnant women was 1.9-3.1%. While the reference values of HbF in males was 0.0-0.0%, in females was 0.0-0.9%, and in pregnant women was 0.0-4.3%. Approximately 2.3% cut-off points for pregnant women was determined to be optimal for α-thalassemia screening. In the entire group, 2.5% was best for all α-thalassemia screenings. The cut-off for β-thalassemia screening using HbA2 was 3.2% for the entire group. The reference interval of HbA2 for pregnant females group was significantly lower than other groups. Therefore, we recommend cut-off points of HbA2 for α-thalassemia at 2.3% for pregnant women. While partitioning was not needed due to gender. Gender and pregnancy had little effect on the cut-off points of HbA2 for β-thalassemia carrier.

Accurate detection of α-globin gene copy number variants with two reactions using droplet digital PCR

ABSTRACT Background The α-thalassemia is a highly prevalent disease in tropical and subtropical regions, including southern China, and is mainly caused by deletion in α-globin genes (HBA1 and HBA2). The clinical manifestation of α-thalassemia is highly correlated with the copy number of α-globin genes. The decrease in copy number results in α-thalassemia, while duplication or triplication compounded with β-thalassemia may aggravate the clinical manifestation. However, the common methods used to measure the copy number variants can only detect the three common types: –SEA, -α3.7, and -α4.2, and may easily miss the rare deletional type and duplication or triplication cases. Therefore, a new method that allows the detection of different copy number variants in α-globin genes simultaneously and accurately needs to be established. Methods A total of 428 peripheral-blood and fetal chorionic villus or amniotic fluid samples were used in this study. We employed a pair of primers and two probes, one for HBA1 and another for HBA2, to perform droplet digital polymerase chain reaction (ddPCR). Each reaction needed the ddPCR of RPP30 as a reference gene to calculate the copy number. Results We accurately detected the copy number variants in α-globin genes, including the common form α-thalassemia, triplications such as αααanti4.2, and trisomy 16, by performing only two reactions. The accuracy rate for detecting the copy number of α-globin genes was up to 100%. Conclusion In conclusion, ddPCR served as an accurate and rapid method for detecting copy number variations in the clinical screening for α-thalassemia.

Hb Bart's Quantitative Analysis in the Screening of α-Thalassemia

To research the relationship between difference types of α-thalassemia gene types and Hb Bart's hemoglobin bands. Capillary electrophoresis was used to screen thalassemia gene for the newborn form January 2020 to December 2020, and the thalassemia gene was detected by PCR or PCR-NGS in the positive patients. The relationship between α-thalassemia gene and Hb Bart's hemoglobin was compared and analyzed statistically. There were significant differences in Hb Bart's hemoglobin among the different α-thalassemia mutation types, Hb Bart's was the highest in --SEA/-α3.7 compound heterozygous mutation, then in --SEA/αα single heterozygous deletion type and in -α3.7/-α3.7，-α3.7/-α4.2compound heterozygous mutation, and in αqsα/αα, αcsα/αα single heterozygous point mutation, least in -α3.7/αα and -α4.2/αα single heterozygous deletion type. There were significant difference among the each groups. The Hb Bart's content of different genotypes of α-thalassemia are significantly different. The Hb Bart's content shows high application value in α-thalassemia screening and genotyping identification.

Erythrocyte Indices and Hemoglobin Analysis for α-Thalassemia Screening in an Area with High Carrying Rate.

Carriers of α-thalassemia exhibit hypochromic microcytosis with mean corpuscular volume (MCV) < 80fL, mean corpuscular hemoglobin (MCH) < 27pg, and reduced hemoglobin A2 (HbA2). We studied the distribution and diagnostic efficiencies of these indicators and their combinations in patients with and without alpha-thalassemia. Based on genetic diagnosis, 10,883 participants were divided into alpha-thalassemia group (n = 1655) and negative-for-alpha-thalassemia group (n = 9228). Erythrocyte parameters and hemoglobin analysis of the groups were analyzed. Moreover, we compared the four screening schemes (MCV/MCH, MCV/MCH/HbA2, MCV + MCH, MCV + MCH + HbA2) to find the best for α-thalassemia screening. The genotypes of --SEA/αα, and -α3.7/αα are the most prevalent with 54.9% and 27.6% in Fujian Province, China. There were significant differences in the distribution of MCV, MCH, and HbA2 in the two groups. Among the three, MCH exhibited the highest sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy. Although the four screening schemes have their advantages, there are significant differences in their sensitivity and specificity. MCV + MCH had the best diagnostic performance (72.6% sensitivity, 89.0% specificity) as well as the highest Youden index (61.59%). Our results showed that MCH could be used to screen α-thalassemia instead of MCV and HbA2. However, it is recommended that MCV/MCH/HbA2 screening be used in areas with high α-thalassemia incidence to increased sensitivity.

Values of MCV,MCH,ROFT and HbA2 for Screening α-Thalassemia in Guangdong Area

To investigate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell osmotic fragility test(ROFT) and hemoglobin A2(HbA2) in screening of α-thalassemia in Guangdong area. A total of 285 peripheral blood samples in patients treated in our hospital from January 2017 to December 2017 were collected. The detection of thalassemia gene was used as the gold standard, while blood routine examination, hemoglobin electrophoresis, and red cell osmotic fragility test were simultaneously performed. The optimal cut-off values in MCV, MCH, ROFT and HbA2 in α-thalassemia were determined by receiver operator characteristic curve (ROC curve). The most common types of α-thalassemia gene was --SEA/αα (54.59%). Compared with the control group, the differences in MCV, MCH, ROFT and HbA2 showed statistically significantce between different types of α-thalassemia (P<0.05). The best cut-off values of MCV, MCH, ROFT, and HbA2 in the diagnosis of α-thalassemia were 81.45 fl, 27.35 pg, 79.95%, and 2.55% respectively. For different laboratories, the cut-off values need to be established for screening α-thalassemia suitable in their own local region．The values of MCV, MCH, ROFT and HbA2 shows higher accuracy and sensitivity in the diagnosis of α-thalassemia. It is recommended to use MCV<81.45fl, MCH<27.35 pg, ROFT<79.95% and HbA2<2.55% as the standards for screening α-thalassemia in Guangdong area.

Clinical Significance of Screening Thalassemia with High- Throughput Sequencing

To evaluate the diagnostic value of high-throughput sequencing (NGS) in screening the thalassemia genes. The peripheral blood of 2 858 cases of pre-pregnancy and pregnancy from October 2014 to October 2016 randomly were collected in department of obstetrics, the third people's hospital in Dongguan city. Peripheral blood was used for the blood routine examination, hemoglobin electrophoresis, traditional thalassaemia gene screening and NGS. The rate of missed diagnosis for α-thalassemia and β-thalassemia using NGS was 0.87% and 1.59%, respectively. Meanwhile, the missed rate of screening for α-thalassemia and β-thalassemia by traditional screening models was 26.77% and 2.38%, respectively. The area under the ROC curve of α-thalassemia and β-thalassemia screened by NGS was 0.994 and 0.991, respectively, however, the area under the ROC curve of screening for α-thalassemia and β-thalassemia by the traditional screening model was 0.866 and 0.988, respectively. The sensitivity, rate of missed diagnosis, Youden index and negative predictive value of screening for α-thalassemia and β-thalassemia using NGS all were superior to those using traditional screening. Compared with the traditional screening model, the NGS screening for thalassemia genes shows a high accuracy, moreover can avoid missed diagnosis resulted from screening by conventional method, suggesting that the NCS possesses the accurate and diagnostic value for screening of thalassemia and can widely apply to clinical practise so as to provid the guarantee for early diagnosis of thalassemia.

Values of Hematological Indicators in the Screening of α-Thalassemia in Fujian Area of China

To analyze the genotypes and the hematological phenotypic characteristics of α-thalassemia in different areas of Fujian and to evaluate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (Hb), RBC distribution width/red blood cell (RDW/RBC) for screening α-thalassemia in this area. The Gap-PCR assay was applied for detecting 3 common deletional mutations of patients with α-thalassemia, and the reverse dot-blot (RDB) assay was adopted to detect the foci of 3 common non-deletional gene mutations.Then,the hematological parameters of individuals with α-thalassemia were analyzed. Finally, the optimal cut-off value in hematological indexes for screening α-thalassemia were determined by the ROC curve. Altogether 16 types of gene mutations were found in 772 patients with α-thalassemia. Among them, the -SEA/αα deletion mutation was the most common which was observed in 521 cases(67.49%). Compared with the control group, the differences in MCV, MCH, and Hb were statistically significant between the patients of the same sex but no same type. In male groups, the RDW/RBC ratio was statistically significant in individuals of light type and HbH disease as compared with the healthy control group. But in female groups, the statistical different of RDW/RBC ratio was found between only HbH disease group and control group. MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L, with the highest specificity and the highest sensitivity, were the best cut-off points for screening α-thalassemia in the laboratory. Due to the difference of regional heterogeneity and hospital equipment environment, the different laboratories need to establish cut-off value for screening α-thalassemia suitable for its local region. In future, our laboratory can use MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L for value for clinical screening, of α-thalassemia.

A Novel α-Thalassemia Screening Scheme in Neonates with Higher Sensitivity and Specificity Using a Combination of Hb Bart's Quantity and Red Blood Cell Indices

Background: Besides detecting hemoglobin (Hb) variants such as sickle cell disease for early preventive measure, newborn screening (NBS) program using Hb profile could identify other forms of thalassemia (thal) and Hb disorders. Due to continuous expression of fetal Hb (γ globin chains), the presence of Hb Bart's (γ4) at birth caused by unbalanced α to β like-globin synthesis have been used to screen for α-thal. So far, this NBS approach appears to be the only laboratory evaluation of α-thal screening for both carriers and Hb H disease since soon after birth Hb Bart's level would decline and we can only use DNA analysis to identify α-thal traits. Neonatal screening of αº-thal due to two missing α-globin genes (–/αα or -α/-α) from normal four copies and Hb H disease (3 α-globin genes lost; –/-α) could be achieved by using the percentage of Hb Bart's cutoffs from isoelectric focusing (Jindatanmanusan P et al. 2014) or capillary electrophoresis (Suksangpleng T et al. ASH 2017). However this approach has a major limitation to screen for α+-thal trait both deletional (-α/αα) and non-deletional (αTα/αα) types due to rather low sensitivity and specificity.Objective: To develop a novel scheme for early detection of all types of α-thal using a combination of neonatal hematological parameters and Hb profiles at birthMethods: After informed consent approved by the local ethical committee, 0.3-ml EDTA whole blood samples and dried blood spot were collected within 48 hrs after birth in newborn delivered at Siriraj Hospital by heel prick puncture. We measured the complete blood count (CBC) by using Unicel® DxHTM800 Coulter® Cellular Analysis System (Beckman Coulter, Brea, USA). The gDNA was also extracted for PCR-based molecular analysis to detect > 98% of α- and β-globin mutations mostly found in Thailand. Neonatal Hb profile was measured in qualitative and quantitative analysis using Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). All data from each genotype groups were statistically analyzed using PASW statistics 18 Software (SPSS Inc, 2015).Results: Total 732 healthy Thai neonates consisted of normal α-globin alleles (αα/αα; n=505, 69%), α+-thal carrier (-α/αα; n=139, 19%), non-deletional α-thal carrier (αTα/αα, n=33, 4.5%), α°-thal carrier (–/αα) and homozygous α+-thal (-α/-α; n=43, 5.9%), compound heterozygous α+-thal with non-deletional type (-α/αTα; n=8, 1%), and Hb H disease (–/-α; n=4, 0.6%). This population had normal β-globin genotype (ββ; n=533, 72.8%), Hb E carrier (βEβ; n=183, 25%) and homozygous Hb E (βEβE; n=16, 2.2%). Interestingly, red cell indices (MCV and MCH) and Hb Bart's levels were significantly altered due to different α-globin genotypes but not by Hb E coinheritance, gender, gestational age or history of neonatal jaundice. These suggested that the variation of red cell indices at birth is largely dependent on the number of expressed α-globin genes. Therefore we hypothesized that by using a combination of red cell indices and Hb Bart's level, we could improve the efficiency of α-thal screening at birth. Indeed, we have identified several cutoffs of Hb Bart's, MCV and MCH by using a ROC analysis that could differentiate individuals with different defective α-globin genes (data not shown). We have developed a range of cutoffs and in combination using either MCV or MCH as a first screening tool followed by Hb Bart's levels, the sensitivity and specificity of α-thal screening could significantly increase (Figure 1A and B). Moreover, we previously showed the 30% sensitivity and 94% specificity to detect α+-thal trait by using Hb Bart's cutoff alone. Interestingly, our approach can increase both sensitivity and specificity up to >90%. This was also true for other types of α-genotypes (Table 1). This novel approach has a significant impact on screening for both deletional and non-deletional α+-thal traits (-α and αTα).Conclusion: For the first time, this novel combined screening using either MCV or MCH together with Hb Bart's could be used to detect all types of α-thal with a very high sensitivity (>85%) and specificity (100%). This approach would be useful to identify individuals with α-thal as a part of our national program for prevention and control of thalassemia disease currently operating in Thailand. Moreover our recommended screening flow described herein would be useful for other Southeast Asian countries where more than 30% of population is affected by α-thal. [Display omitted] DisclosuresViprakasit:Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding.

Molecular Characteristics of Hb New York [β113(G15)Val→Glu, HBB: c.341T>A] in Thailand

Hb New York or Hb Kaohsiung [β113(G15)Val→Glu (GTG>GAG), HBB: c.341T>A] has been considered a rare β hemoglobin (Hb) variant found originally in an Iranian woman and later in diverse populations but its genetic origin has not been elucidated. Here we report molecular and hematological descriptions of this variant found in the Thai population. Among 5643 subjects referred for hemoglobinopathy investigation during January 2015 to September 2017, 183 (3.2%) were found to carry several Hb variants, including β chain variants (n = 135, 2.4%), α chain variants (n = 33, 0.6%), Hb Lepore-Hollandia (NG_000007.3: g.63290_70702del) and Hb Lepore-Boston-Washington (NG_000007.3: g.63632_71046del) (δβ hybrid Hb) (n = 12, 0.2%) and δ chain variants (n = 3, 0.05%). Of patients with β chain variants, six with normal high performance liquid chromatography (HPLC) patterns, had an abnormal Hb in zone 11 of capillary electrophoresis (CE), the amounts of which ranged from 29.6-45.4% with normal levels of Hb A2 and Hb F. DNA analysis identified a heterozygous Hb New York mutation in all cases. Further screening of α-thalassemia (α-thal) identified coinheritance of α+- and α0-thal in two of them who had reduced levels of Hb New York. Haplotype analysis suggested that the Thai Hb New York was likely associated with a single β-globin haplotype [+ – – – – + +], indicating that it was of the same origin. Hematological findings and simple DNA assay based on allele-specific polymerase chain reaction (PCR) for rapid detection of Hb New York are presented.

Thalassemia Screening Using Different Automated Blood Cell Counters: Consideration of Appropriate Cutoff Values.

To evaluate the use of MCV and MCH cutoffs for thalassemia screening in areas with a high frequency of thalassemia and hemoglobinopathies using different hematology analyzers. Blood samples of known α(0)-thalassemia and β-thalassemia carriers were analyzed to establish the appropriate cutoffs for each analyzer. These selected cutoffs were validated prospectively for screening of α-thalassemia, β-thalassemia in combination with the dichlorophenolindophenol test for hemoglobin E on another 288 Thai and 325 Laotian students. Genotypes were defined by standard hemoglobin and DNA analyses. The appropriate cutoffs for the Sysmex XS-800i and Sysmex XN-1000, Coulter LH 780, and Pentra ES-60 were found to be 78 fL for MCV and 25 pg for MCH. These were 82 fL and 25 pg for the Cell-Dyn Ruby. Further validation on Thai and Laotian students revealed 100% sensitivity and specificity of higher than 80% for all analyzers. While using MCV in screening requires establishment of appropriate cutoffs, MCH appears to be applicable to all analyzers. Each analyzer should be evaluated and appropriate cutoffs should be established before application in the field.

Comparison of three different α-thalassemia prenatal screening strategies used in Guangdong, China

Objective To compare the effect and cost of three different α-thalassemia prenatal screening strategies used in Guangdong, China, and to provide evidence for α-thalassemia prevention. Methods In total, 13 284 hospital-delivery couples and 13 369 newborns/fetuses (offspring) from 21 counties or districts of Guangdong Province were included in this study, who were treated from June to December 2012. Mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A2 (Hb A2) were detected in the couples, and 6 types of α-globin gene mutations were found in all couples and newborns. The strategies were MCV/MCH and serum Hb A2 (protocol Ⅰ) or parallel screening based on pregnant women (protocol Ⅱ), and serum screening based on couples (protocol Ⅲ). The validity and reliability of the three strategies were then compared using the Chi-square test. Results The sensitivity and the specificity of pregnant women who were α-thalassemia carriers in protocolⅠ were 74.82% (1 352/1 807) and 74.11% (8 506/11 477), and were 89.82% (1 623/1 807) and 48.60%(5 578/11 477) in protocol Ⅱ, respectively. And 1.67% (221/13 284) couples were both α-thalassemia carriers by the gene test. The rate of missed diagnosis in both α-thalassemia carrier couples in protocolsⅠ,Ⅱ andⅢ was 50.68% (112/221), 11.76%(26/221) and 11.31%(25/221), respectively. In couples who needed prenatal diagnosis, the rates of missed diagnosis, sensitivity, specificity, positive predictive value, and negative predictive value were 17.46%(11/63), 82.54%(52/63),98.35% (13 003/13 221), 19.26%(52/270) and 99.92 %(13 003/13 014) in protocol Ⅰ; 4.76%(3/63), 95.24%(60/63), 88.18%(11 658/13 221), 3.70%(60/1 623) and 99.97%(11 658/11 661) in protocolⅡ; and 3.17%(2/63), 96.83%(61/63), 59.31%(7 842/13 221), 1.12%(61/5 440) and 99.97%(7 842/7 844) in protocolⅢ, respectively. The diagnosis of severe α-thalassemia was not missed in all three screening strategies. The mean cost of protocols Ⅰ, Ⅱ and Ⅲ for detecting a couple who needed prenatal diagnosis was 37 049.23, 50 836.00 and 40 321.64 RMB, respectively. Conclusions The three screening protocols have good efficiency in screening for α-thalassemia. However, protocols Ⅱ and Ⅲ are preferred when financial conditions permit. Key words: Alpha-thalassemia; Prenatal diagnosis; Genetic testing

Validation of the immunochromatographic strip for α-thalassemia screening: a multicenter study

α(0)-Thalassemia occurs from a deletion of 2 linked α-globin genes and interaction of these defective genes leads to hemoglobin (Hb) Bart's hydrops fetalis, the most severe and lethal thalassemia syndrome. Identification of α(0)-thalassemia carriers is thus essential for the prevention and control program. An immunochromatographic (IC) strip test was developed for rapid screening of α(0)-thalassemia by testing for Hb Bart's in the blood samples using a specific monoclonal antibody against Hb Bart's. To evaluate its sensitivity and specificity, the IC strip test was assessed in a cohort with various thalassemia genotypes from 4 different laboratories in Thailand and Australia. The result showed 97% sensitivity in α-thalassemia carriers with 2 α-globin genes deletion and Hb H disease. This is, in particular, the useful rapid screening test for regions where β-thalassemia and homozygous Hb E are also common. Similar hematologic and Hb data make it impossible to address the concomitant inheritance of α(0)-thalassemia in these samples without polymerase chain reaction (PCR)-based techniques, leading to misdiagnosis of the risk of having Hb Bart's hydrops fetalis. However, α-globin genotyping should be carried out in samples with positive IC strip as positive reactivity was also observed in homozygous α(+)-thalassemia carriers who have 2 trans α-globin gene deletions. These results indicate that in combination with red blood cell indices, the IC strip test could rule out mass populations for further α(0)-thalassemia detection by PCR-based analysis. The Alpha Thal IC strip also has the potential to replace testing for Hb H inclusion bodies, as it appears to be more sensitive, specific, and less labor intensive.

A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia

The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. Arapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.

The value of MCV, MCH and HbA(2) in laboratory screening of thalassemia

To explore the roles of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A(2) (HbA(2)) in the laboratory screening of thalassemia, and to find optimal screening modality for different conditions. From September 2008 to May 2011, 1384 subjects underwent thalassemia screening at Department of Obstetrics and Gynecology of Nanfang Hospital. Of them, 1036 cases were diagnosed with thalassemia (408 α-thalassemia, 608 β-thalassemia, and 20 αβ compound thalassemia, thalassemia group) and 348 without thalassemia, non-thalassemia group. All subjects were screened respectively for MCV, MCH and HbA(2). Analyses were performed in all subjects to assess the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy respectively associated with MCV, MCH and HbA(2) alone, combination of MCV and MCH, and combination of MCV, MCH and HbA(2). (1) In the thalassemia group, the sensitivity of MCV alone was 92.9% (379/408) for α thalassemia, 99.3% (604/608) for β thalassemia and 100.0% (20/20) for αβ compound thalassemia. In the non-thalassemia group, the specificity of MCV alone was 75.0% (261/348). (2) In the thalassemia group, the sensitivity of MCH alone was 92.9% (379/408) in α thalassemia, 99.0% (602/608) in β thalassemia and 100.0% (20/20) in αβ compound thalassemia. In the non-thalassemia group, the specificity of MCH alone was 72.7% (253/348). (3) The sensitivity of Hb A(2) alone was 67.4% (275/408) for α thalassemia, 97.5% (593/608) for β thalassemia, and 100% (20/20) for αβ compound thalassemia while it's specificity was 72.4% (252/348) in the non-thalassemia group. (4) With positive indexes of MCV, MCH and MCV + MCH, when HbA(2) > 3.5% it had a high value in β-thalassemia screening, but when HbA(2) < 2.5% it had little value in α-thalassemia screening. (5) As a single marker, MCV and MCH had better sensitivity, specificity, positive predictive value, negative predictive value and diagnosis accuracy than HbA(2). MCV + MCH was the best for overall screening, but for β thalassemia screening, MCV + MCH + HbA(2) was the best. MCV and MCH are suitable for epidemic screening in a large population, physical examination and premarital check-up. Hb electrophoresis and thalassemia gene diagnosis are recommended for subjects with positive MCV and MCH indexes. Diagnoses of α and β-thalassemia gene are recommended for pregnant women with positive MCV and MCH indexes.

Detection of α-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments

The aim of this study was to establish a sensitive method that can detect the presence of not only the common but also the unusual or unknown α-globin gene deletions for screening of α-thalassemia. We used quantitative multiplex PCR of short fluorescent fragments (QMPSF) for the α-globin genes (HBA) to screen α-thalassemia deletions. We set up and validated HBA-QMPSF using 50 negative and 100 positive controls of deletional α-thalassemia. To evaluate its ability to detect the presence of the common and unusual or unknown α-globin gene deletions, 579 unrelated samples were simultaneously analyzed using this assay and multiplex Gap polymerase chain reaction (Gap-PCR). The inconsistent results were further confirmed by multiplex ligation-dependent probe amplification (MLPA). HBA-QMPSF was capable of detecting α-globin gene deletions with an acceptable variability as shown by mean values (SD) of allele dosage for the heterozygous deleted control obtained from intra- and inter-experimental replicates [0.63 (0.01) and 0.61 (0.03)]. In 572 out of the 579 unrelated subjects, HBA-QMPSF and multiplex Gap-PCR gave consistent results. In seven cases which were finally proved to be composed of one rare deletion--Thai/-α3.7, one novel deletion--SEA/-α2.8, four αααanti3.7/αα and one αααanti4.2/αα triplications, HBA-QMPSF showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to give the correct diagnosis. HBA-QMPSF is able to detect the presence of the common and unusual or unknown α-thalassemia deletions and duplications. It can be used as an initial screening test for α-thalassemia caused by HBA gene copy number alteration.

Diagnostic Utility of Isoelectric Focusing and High Performance Liquid Chromatography in Fetal Cord Blood Screening for Thalassemia and Non-Sickling Hemoglobinopathies,

Abstract 4202 Background:Thalassemia and non-sickling hemoglobinopathies are highly prevalent in Southeast Asia. High performance liquid chromatography (HPLC), β-thalassemia short program, is the most common technique routinely used in this region, while IEF is rarely employed. However, HPLC, β-thalassemia short program, cannnot quantify HbBart’s, as well as HbH. In addition, the acetylated HbF, which is frequently found in cord blood, interferes with the HbBart’s and HbH peaks on HPLC. We primarily aim to compare diagnostic utilities of IEF and HPLC in fetal cord blood screening for prenatal diagnosis of thalassemia and non-sickling hemoglobinopathies in Thailand. Methods:One-hundred and two cord bloods from the National Cord Blood Bank and fetuses of high risk couples were analyzed using IEF and HPLC, β-thalassemia short program, and correlated with red cell indices and DNA analyses. HbBart’s was quantified only on the IEF method. The sensitivity and specificity to screen for α-globin and β-globin gene mutations were determined. Results:Of 88 cases showing HbBart’s on hemoglobin analysis, IEF yielded 98.9% sensitivity to identify at least one α-globin gene defect (−α−3.7, -α−4.2, -αCS, -αPS, –SEA, –Thai, -α−3.7/-α−3.7 and -α−3.7/-αCS), while HPLC yielded only 83.7% sensitivity to detect at least one α-globin gene defect. All missing cases on HPLC were single α-globin gene deletion that had HbBart’s less than 1.5% of total hemoglobin measured on IEF. Hb Constant Spring (CS) and Hb Pakse (PS) heterozygotes (N = 11) had small amount of HbBart’s less than 3%, but neither HbCS nor HbPS was detectable on both tests. HbBart’s ≥ 2% yielded 100% sensitivity to identify two α-globin gene defects, while HbBart’s ≥ 3% yielded 100% specificity to diagnose two α-globin gene defects. MCV and MCH are the most helpful red cell parameter in screening for α-thalassemia. MCV ≤ 95 fL yielded 100% sensitivity to identify two α-globin gene deletions, while MCV ≤ 90 fL yielded 100% specificity to identify two α-globin gene deletions. In addition, MCH ≤ 30 pg yielded 100% sensitivity and 100% specificity to identify two α-globin gene deletions.Thirty two cord bloods showed HbA2/E, ranging from 1.9–15.6%. DNA analysis using a reverse dot blot technique for detecting common β-thalassemia mutations in Thailand demonstrated HbE mutation in all cases, while no β-thalassemia mutations were identified. Therefore, detectable HbA2 in cord blood usually represents HbE, not HbA2. Three HbE heterozygotes with two α-globin gene deletions demonstrated high HbBart’s and low MCV, as well as low MCH, similar to those with two α-globin gene deletions alone. On the other hand, HbE heterozygotes with single α-globin gene defects could not be differentiated from pure HbE heterozygotes. Conclusion:IEF is superior to HPLC β-short program in screening for α-globin gene defects in prenatal cord blood analysis. The amount of HbBart’s, MCV and MCH are helpful for identifying α-thalassemia. The presence of HbA2 in cord blood indicates HbE mutation, but not β-thalassemia. Disclosures:Sutcharitchan:Novartis: Research Funding.