Microcytic anemia in children: parallel screening for iron deficiency and thalassemia provides a useful opportunity for thalassemia prevention in low- and middle-income countries.

Microcytic anemia in children is commonly attributed to iron deficiency without attempting to find the cause. Inadequate investigations to exclude hemoglobinopathies lead to missed opportunities for identification of thalassemia carriers. Here we aim to describe the relative contribution of iron deficiency and thalassemia to microcytic anemia in children. This hospital-based prospective study was conducted at the Colombo North Teaching Hospital, Ragama, Sri Lanka. All newly diagnosed patients with microcytic anemia were recruited and data were collected using an interviewer-administered questionnaire. Full blood count, blood film, serum ferritin, c-reactive protein, quantification of hemoglobin sub-types and α-globin genotype were performed using 4 ml of venous blood. A total of 104 children (Male- 60.5%) were recruited. Iron deficiency was the cause for anemia in 49% whilst 16% and 10% had α- and ß-thalassemia trait respectively. Seven (6.7%) children had co-existing iron deficiency and thalassemia trait while two coinherited α- and ß-thalassemia trait. Children with ß-thalassemia trait had significantly higher red cell count and lower mean corpuscular volume compared to children with iron deficiency. However, none of the red cell parameters were significantly different between children with α-thalassemia trait and iron deficiency. Iron deficiency contributes only to half of children with microcytic anemia; one-fourth had thalassemia trait. Co-existence of iron deficiency and thalassemia trait or co-inheritance of α- and ß-thalassemia trait were found in 9%. Parallel investigation of children with microcytic anemia to diagnose iron deficiency and thalassemia provides an opportunity to identify thalassemia carriers which is beneficial for thalassemia prevention.

Prevention of Hb Bart's (γ4) Disease Associated with the - -(THAI) α(0)-Thalassemia Deletion in Mainland China.

α-Thalassemia (α-thal) is a common inherited disease in southern China. The severest form is Hb Bart's (γ4) disease, in which the affected fetuses almost always die in utero or shortly after birth, and the mothers are at high risk for severe morbidity. The most common type of α(0)-thalassemia (α(0)-thal) in southern China is Southeast Asian (- -(SEA)) deletion. Occasionally, Hb Bart's disease, caused by a compound heterozygosity for the - -(SEA) and - -(THAI) α(0)-thal deletions, can also be encountered. In this study, we report our experience with the prevention of Hb Bart's disease associated with the - -(THAI) α(0)-thal deletion. A total of 385 couples at risk for Hb Bart's disease, including seven who tested positive for the - -(SEA) deletion in one partner and the - -(THAI) deletion in the other, were found. Different prenatal procedures were offered, depending on the gestational age at presentation. Sixty-six affected fetuses were diagnosed prenatally; among these, two cases of Hb Bart's disease were compound heterozygotes for the - -(SEA) and - -(THAI) deletions. All affected pregnancies were terminated in time. We also presented a diagnostic protocol for identification of α(0)-thal trait that can reduce the number of samples for detection of the - -(THAI) deletion.

Molecular Diagnosis of α°-Thalassemia Through Urine DNA: A Novel DNA Source to Facilitate Prevention Programs in Remote Geographical Areas.

We assessed whether urinary DNA sediment was a feasible sample type for the molecular diagnosis of α-thalassemia (α-thal) mutations. Urine samples (5-10 mL) were collected from 218 male and female volunteers. The cells were centrifuged, and DNA was isolated according to the protocol of a commercial DNA isolation kit. Detection of the α(0)-thal [Southeast Asian (- -(SEA)) and - -(THAI)] deletions was performed using quantitative real-time polymerase chain reaction (q-PCR), in addition to conventional gap-PCR. The results revealed that DNA extracted from urinary sediment presented an average DNA content of 11.2 ± 5.5 ng/µL, and the 260/280 ratio indicative of DNA purity, was 1.2 ± 0.2. The overall q-PCR threshold cycle was 31.2 ± 2.3. The melting temperature for the - -(SEA) deletion was 87.3 ± 0.1 °C, while that of the wild type sequence was 92.5 ± 0.2 °C. There were 16 (7.3%) α(0)-thal SEA genotypes detected. These results were in agreement with those of the conventional gap-PCR and blood DNA analyses. Thus, DNA from urinary sediment can be efficiently used for the molecular diagnosis of α(0)-thal mutations. This approach allows for rapid diagnosis, is non invasive, and could be useful for preventing Hb Bart's (γ4) hydrops fetalis syndrome.

Problems in determining thalassemia carrier status in a program for prevention and control of severe thalassemia syndromes: a lesson from Thailand.

BACKGROUND: Prevention and control of severe ß thalassemia by carrier detection and identification of couples at risk in developed countries is one of the most successful stories in modern medicine. Similar programs in developing countries especially Southeast Asia, are more problematic because both α and ß thalassemias are highly prevalent. In Thailand, there are limited data on whether we could determine, based on hematological phenotypes, the mutation severity and/or coinheritance of α thalassemia in ß thalassemia traits. METHODS: Comprehensive molecular, hematology and hemoglobin analyses of the α and ß globin genes were performed in 141 healthy individuals identified as ß thalassemia carriers. RESULTS: Seventeen different ß globin mutations were successfully identified out of all cases analyzed. Although the majority of the mutations identified were the ß° or severe ß⁺ thalassemia alleles, a high proportion of mild mutations (25%) was observed. Of these ß thalassemia traits, 22.3% were found to co-inherit the α thalassemias. Milder hematological phenotypes were noted in ß⁺ compared with ß° thalassemia traits when the α globin genes were intact. Although co-inheritance of α° thalassemia might be suspected in cases with skewed profiles, due to the overlapping values, it remains difficult to apply these parameters for reliable carrier determination. CONCLUSIONS: A combination of hemoglobin analysis and DNA testing seems to be the best way to confirm carrier status in a region with high frequency for both α and ß thalassemias. Underdiagnoses of carrier status could hamper the effectiveness of a thalassemia prevention and control program.

Prenatal control of nondeletional α-thalassemia: first experience in mainland China.

OBJECTIVE: To demonstrate the performance of nondeletional α-thalassemia prevention at a mainland Chinese hospital. METHODS: A prenatal control program for nondeletional hemoglobin H (Hb H) disease was conducted from January 2010 to June 2012. All couples were screened for α-thalassemia trait, and for couples in whom one partner was tested positive for α(0) -thalassemia, the other was subjected to screening for Hb Constant Spring and Hb Quong Sze mutations. Prenatal diagnoses were offered in pregnancies of couples at-risk for nondeletional Hb H disease. RESULTS: Of the 30,152 couples screened, 18 (0.06%) were diagnosed as at risk for nondeletional Hb H disease. There were other 13 at-risk couples who were referred to prenatal diagnosis because they had previously an affected child. Of the 31 cases with prenatal invasive tests, 11 (35.5%) had diagnosis by chorionic villous sampling, and 20 (64.5%) had amniocentesis. Totally, 12 fetuses were diagnosed with nondeletional Hb H disease, and all of the affected pregnancies were terminated. CONCLUSION: Implementation of a prevention and control program accompanying with a referral system for prenatal diagnosis is technically feasible in southern China, and a number of nondeletional Hb H disease have been prevented during the past 3 years of operation.

Preimplantation genetic diagnosis of alpha-thalassemia-SEA using novel multiplex fluorescent PCR.

PURPOSE: Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis (PND) giving couples at risk a chance to start a pregnancy with a disease-free baby. This study aimed to develop a new PGD protocol for alpha-thalassemia(-SEA) mutation, the commonest Mendelian disorder. PATIENTS AND METHODS: Multiplex fluorescent PCR was employed for mutation, contamination and linkage analysis. A couple experienced termination of pregnancy following positive PND decided to join the project. RESULTS: Novel primers for alpha-thalassemia(-SEA) mutation amplifying 5 DNA fragments were developed. Two PGD cycles were performed, resulting in an un-affected baby. PND confirmed the heterozygous result. From 24 embryos, 87.5% of affected genotype were of best quality compared to 0% and 18.2% of those with normal and heterozygous, respectively. CONCLUSIONS: A novel PCR protocol for the common alpha-thalassemia(-SEA) mutation is reported. This test should be widely applicable. Interestingly, a potential effect of alpha-thalassemia(-SEA) mutation on preimplantation embryonic development was noticed.

[Large-scale population-based genetic screening and prenatal diagnosis for thalassemias in Zhuhai City of Guangdong Province].

OBJECTIVE: To report the results of preventive control program of severe thalassemias in Zhuhai City of Guangdong Province from 1998 to 2010. METHODS: As the guide centre of marriage and childbearing and the greatest maternity hospital in Zhuhai City of Guangdong Province, Zhuhai Municipal Maternity and Child Healthcare Hospital constructed the genetic screening network for thalassemias testing and referred for follow-up and for genetic counseling. The couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled to this preventive control program. A conventional strategy of screening for heterozygote was used to identify the α- and ß-thalassemia traits in women and their spouses according to the standard procedures of hematological phenotype analysis which was recommended by Thalassemia International Federation (TIF). Then those suspected couples at risk were diagnosed for α- and ß-thalassemia by PCR-based DNA assays. The couples at risk for severe thalassemias were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus in the rights of consent and of option voluntarily. RESULTS: From January 1998 to December 2010, 85 522 brides and grooms-to-be for premarital screening and 41 503 pregnant women in addition to 14 141 partners for prenatal screening were recorded, the covering rates of premarital screening and prenatal screening in the city were 92.698% (from 1998 to 2003) and 27.667% (from 2004 to 2010), respectively. Totally 10 726 cases were found to be the carriers of thalassemias, with 7393 for α-thalassemia (5.237%, 7 393/141 166) and 3333 for ß-thalassemia (2.361%, 3 333/141 166). A total of 257 couples at-risk for severe thalassemias were detected including 190 for α-thalassemia and 67 for ß-thalassemia. Among them, 251 (97.7%, 251/257) couples were performed prenatal diagnosis. During the preventive control program, a total of 72 fetuses with severe thalassemias including hemoglobin H disease were voluntarily terminated. In Zhuhai City, the average annual birth rate of fetuses with severe thalassemia was declined by 32.9% (49/149). CONCLUSIONS: This study has reduced effectively birth rate of perinatal infants with severe thalassemias in Zhuhai City by genetic screening and prenatal diagnosis of thalassemia in the large population of 13 years. Our summary comes out of technical proposals for prenatal screening and diagnosis, which could be take example by preventative control of thalassemia in other regions of China where are prevalent.

High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.

OBJECTIVE: To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1. MATERIALS AND METHODS: Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele]. RESULT: The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished. CONCLUSION: HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination.

Preimplantation genetic diagnosis for alpha-thalassaemia in China.

PURPOSE: To report the usage of PGD for alpha-thalassaemia with the - -(SEA) genotype. METHOD: A PGD protocol using fluorescent gap PCR was performed for 51 cycles on 43 couples with the - -(SEA) genotype. Allele drop-out and amplification failure rates were retrospectively analyzed. RESULTS: A total of 472 embryos were biopsied. Amplification was achieved in 390 blastomeres, accounting for an amplification rate of 82.6%. In total, 120 wild-type, 94 heterozygotes and 140 homozygous mutant embryos were diagnosed. The successful diagnosis rate was 75.0%. The ADO rate in 49 blastomeres from six donated embryos was 16.4%. One hundred and fifty four embryos were transferred, resulting in 25 clinical pregnancies with an implantation rate of 24.0%. CONCLUSIONS: Single-round fluorescent gap PCR is a feasible and effective strategy in the PGD for alpha-thalassaemia with the - -(SEA) genotype.

False positive rates of thalassemia screening in rural clinical setting: 10-year experience in Thailand.

This retrospective study aimed to describe the magnitude of false positive screening for thalassemia in a primary care setting of Thailand. The study was conducted from 1999 to 2008 and analyzed 13,745 positive cases. It involved a combination of one tube osmotic fragility (OF) and dichlorophenol indophenol (DCIP) precipitation tests. The number of cases increased over the ten-year period, corresponding well to an exponential model. Based on hemoglobin and DNA analysis, cases with alpha-thalassemia 1, beta-thalassemia, and hemoglobin E were defined as true positive cases, and the remaining were considered as false positives. The false positive rate was in the range of 20.1-36.1%. The proportion of false positive cases of thalassemia from the screening tests was associated with a trend which was statistically significant (p < 0.001). The estimated cost of further hemoglobin analysis resulting from false positive cases was approximately 40.2-72.2 million THB/year for an estimated 800,000 annual births. The combination of the OF and DCIP test, which has been the strategy for screening of thalassemia and HbE in pregnant women throughout this country, resulted in a large economic burden in terms of high cost and workload associated with further hemoglobin and DNA analyses of false positive samples. Measures to reduce false positive should be developed and implemented.

Alpha-thalassemia: Hb H disease and Hb Barts hydrops fetalis.

alpha-Thalassemia mutations are one of the most common mutations of man, and they cause Hb H disease and Hb Barts hydrops fetalis. Hb H disease is not necessarily a benign disorder as has been generally thought. Furthermore, in southern China and in Southeast Asia, there are 2-3 times more fetuses afflicted with the invariably fatal Hb Barts hydrops fetalis than with the beta-thalassemia major or intermedia. These findings underscore the public health importance of these hereditary disorders, and they call for better education, diagnosis, treatment, prevention, and research for these diseases.

A survey of current United Kingdom practice for antenatal screening for inherited disorders of globin chain synthesis. UK Forum for Haemoglobin Disorders.

AIMS: To document current United Kingdom practice for antenatal screening for inherited disorders of globin chain synthesis and to compare such practice with guidelines published by the British Committee for Standards in Haematology and the Standing Committee on Sickle Cell, Thalassaemia and other Haemoglobinopathies (SMAC). METHODS: The members of the UK Forum on Haemoglobin Disorders were surveyed about their current practice for antenatal haemoglobinopathy screening. The UK Forum is a national group of haematologists, paediatricians, laboratory scientists, and counsellors working in the field of diagnosis and management of disorders of haemoglobin synthesis; such disorders including the alpha and beta thalassaemias, sickle cell disease, and other haemoglobinopathies. RESULTS: Completed questionnaires from 38 hospitals (or cooperating groups of hospitals) were analysed. The great majority of hospitals were applying appropriate laboratory methods, but problems were commonly encountered in ensuring that appropriate testing of antenatal patients and, when necessary, of their partners, was carried out early in pregnancy. When screening was selective there was quite often a failure to identify all women in whom testing was indicated, and cut off points used as an indication for further testing were sometimes inappropriate. CONCLUSIONS: Many practical problems are still encountered in following guidelines for the antenatal diagnosis of haemoglobinopathies. A need for improved administrative procedures and increased funding was identified. In addition there is a need for agreed guidelines giving more specific advice on technical aspects of laboratory practice.

Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype.

We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured. The brilliant cresyl blue test reliably detects couples at risk for hemoglobin Bart's hydrops fetalis. Measurement of the number of inclusion bodies differentiates the alpha-thalassemia genotypes in the absence of a coincident beta-chain synthesis deficiency, such as hemoglobin E or beta-thalassemia. The test appears to identify patients, such as those with the Thai and Filipino deletion variants, whose alpha-thalassemia cannot be definitively characterized by DNA testing when only alpha- and zeta-globin probes are used in the analysis. We also found evidence of elevated serum ferritin levels in many patients with deletion of two or three alpha-globin genes. This study shows that most routine screening for alpha-thalassemia can be performed with three simple tests: (1) the brilliant cresyl blue inclusion study, (2) erythrocyte indices, and (3) iron studies. Analysis with DNA probes is needed in only some circumstances.

Prenatal strategies for reducing severe thalassemia in pregnancy.

OBJECTIVE: To describe the prenatal strategy in reducing new cases of severe thalassemia at Maharaj Nakorn Chiang Mai Hospital. The study design involved a prospective descriptive analysis set in Maharaj Nakorn Chiang Mai Hospital, Chiang Mai University. SUBJECTS: Pregnant women attending antenatal clinic. METHODS: The strategy included: (1) carrier identification by retrospective (history review) and prospective screening program; (2) the couples at risk were counseled and offered cordocentesis; (3) analysis of fetal blood with high performance liquid chromatography (HPLC) or electrophoresis; and (4) counseling for termination of pregnancy in case of affected fetus. The prospective screening consisted of testing for a carrier by a simple erythrocyte osmotic fragility test (EOFT) in women with no risk and testing the husbands of the women with abnormal tests. A pregnancy in which both of the couple were carriers was considered a risk. RESULTS: Cordocentesis was performed in 554 pregnancies at risk, 252 and 302 from retrospective and prospective screening, respectively. Sixty of 252 of the first group had severe thalassemia. In the prospective screening program of 12 680 women, 459 risk couples were identified, 302 pregnancies underwent cordocentesis and 53 (17.5%) had severe thalassemia. This strategy enabled us to identify 113 cases of severe thalassemia (Hb Bart's; 60, beta-thal entities; 53) from 554 cases at risk. CONCLUSION: The strategy proves valuable in the control of severe thalassemia. This extensive experience suggests the strategy be considered an effective way in the control of severe thalassemia in high prevalence areas.

Thalassemia in SouthEast Asia: problems and strategy for prevention and control.

In Southeast Asia alpha-thalassemia, beta-thalassemia, hemoglobin (Hb) E and Hb Constant Spring are prevalent. The gene frequencies of alpha-thalassemia reach 30-40% in Northern Thailand and Laos. beta-Thalassemia gene frequencies vary between 1 and 9%. Hb E is the hallmark of Southeast Asia attaining a frequency of 50-60% at the junction of Thailand, Laos, and Cambodia. Hb Constant Spring gene frequencies vary between 1 and 8%. These abnormal genes in different combinations lead to over 60 different thalassemia syndromes. The four major thalassemic diseases are Hb Bart's hydrops fetalis (homozygous alpha-thalassemia 1), homozygous beta-thalassemia, beta-thalassemia/Hb E and Hb H diseases. The molecular basis of most of these abnormal genes have been recently described. Therefore, it is possible to set a strategy for prevention and control of thalassemia which includes population screening for heterozygotes, genetic counseling and fetal diagnosis with selective abortion of affected pregnancies.

Frequency of alpha-thalassemia-1 of the Southeast Asian-type among pregnant women in northern Thailand determined by PCR technique.

Five hundred pregnant women were analyzed for the presence of alpha-thalassemia-1 of the Southeast Asian (SEA)-type by polymerase chain reaction (PCR) technique at the Maharaj Nakhon Chiang Mai University Hospital in Chiang Mai during the period from April to June 1995. Forty-four of them (8.8%) were recognized as carriers, corresponding to a frequency of 0.044. Homozygous alpha-thalassemia-1 of the SEA-type, the fatal condition of hemoglobin Bart's hydrops fetalis, has an expected frequency of 0.00194, or about 2 hydrops fetalis cases per 1,000 births in this population.

Prevention and control of Hb Bart's disease in Guangxi Zhuang Autonomous Region, China.

OBJECTIVE: To demonstrate the performance of Hb Bart's Disease prevention in Guangxi Zhuang Autonomous Region, China. STUDY DESIGN: A prenatal control program for Hb Bart's disease was conducted from January 2006 to December 2012. A total of 17,555 pregnant women were screened for α-thalassemia in our prenatal screening program. Pregnancy at-risk for Hb Bart's disease was offered the choice of direct invasive testing or the non-invasive approach with serial ultrasonography. RESULTS: A total of 1425 at-risk couples attended the prenatal diagnosis. Three hundred ninety couples were screened at our own hospital, and the remaining 1035 couples were referred from other hospitals. Two hundred and three pregnant women chose non-invasive approach, and 1122 chose invasive testing. A total of 365 fetuses were diagnosed with Hb Bart's disease. All cases were finally confirmed by fetal DNA analysis. Eighty-two cases (22.4%) were diagnosed by chorionic villous sampling and 194 (53.2%) by amniocentesis samples. The other 89 (24.4%) cases were performed by cordocentesis. All of the affected pregnancies were terminated. CONCLUSION: Implementation of a prevention and control program accompanying with a referral system for prenatal diagnosis is technically feasible in Guangxi Zhuang Autonomous Region, China.