Thalassemia and iron deficiency among pregnant women attending antenatal care service at Khao Wong Hospital, Kalasin province

In order to obtain an appropriate control measure for thalassemia and iron deficiency anemia, it is necessary to estimate the health burden in the relevant area. This study aimed to determine the prevalence of thalassemia and iron deficiency in pregnant women attending antenatal care service at Khaowong Hospital, Kalasin province, in which the majority of pregnant women is the “PhuTai” ethnic group. Blood samples taken from 302 pregnant women were investigated for hematological parameters using an automated blood cell counter to diagnose anemia. For the diagnosis of iron deficiency, measurement of ferritin was done by chemiluminescent immunoassay. Hb analysis using cellulose acetate electrophoresis and/or capillary zone electrophoresis was performed to diagnose b-thalassemia and abnormal hemoglobin (Hb).  Alpha-thalassemia genes including Hb Constant Spring (Hb CS) and Hb Pakse’ were identified by the polymerase chain reaction and related technologies. Of the 302 subjects, 36.4 % (95% CI = 31.0-42.1) had anemia, 42.0 % (95 % CI = 32.2-52.3) had iron deficiency and 17.0 % (95 % CI = 10.2-25.8) had iron deficiency anemia. Alpha-thalassemia 1 was found in 9.6% (95% CI = 6.5-13.5), comprising 9.3 % SEA deletion and 0.3 % THAI deletion. The prevalence of a-thalassemia 2 was 27.0 % (95 % CI = 18.6-36.8), comprising 14.0 % 3.7 kb deletion, 1.0 % 4.2 kb deletion and 12 % Hb CS. While 43.7 % (95 % CI = 38-0-49.5) of the subjects were Hb E carriers, only 0.3% (95 % CI = 0.0-1.8) were b-thalassemia carriers. The findings demonstrate the health burden in relation to thalassemia and iron deficiency anemia and indicate a need for appropriate measure to control the disease in this ethnic group.

Performance of thalassemia screening program for identification of at-risk couples at Prayuen Hospital, Khon Kaen province

Complying with national policy of the Ministry of Public Health, Prayuen Hospital has implemented thalassemia screening program since 2006. Based on the operational guidelines, pregnant women attending antenatal care service are screened for thalassemia using OF/DCIP approach. In case of positive results, the husbands are requested to be screened for thalassaemia as well. Accordingly, blood samples of the positive-screened couples are referred to the Regional Medical Sciences Center, Khon Kaen, to investigate further whether they have true risk. In order to evaluate the performance of thalassemia screening program conducted at Prayuen Hospital during 2006-2008, a retrospective data on the number of the first-visit pregnant women, number of positive screened couples as well as number of the at-risk couples were collected. The positive rate of thalassemia screening, the percentage of screened husbands and the positive rate of at-risk couples were calculated. It was found that the rate of positive screened women in 2006, 2007 and 2008 was 53.5 %, 49.3 % and 53.9 %, respectively. Of the positive-screened women, 56.6 %, 76.1 % and 70.8% of the husbands had been screened for thalassemia. During these 3 fiscal years, a total of 260 couples were gathered. Among them, 8 (3.1 %) were at risk of having babies with severe thalassemia diseases, i.e. 6 at risk of b-thalassemia / Hb E and 2 at risk of homozygous a-thalassemia 1. The expense of laboratory identification of at risk couples was 62,500 baht/year (0.9 % of treatment expense for 1 patient). The results demonstrated that Prayeun Hospital had good performance and complied with the goal of the Ministry of Public Health in that at-risk couples were identified. Consequently, the births of new cases with severe thalassemia diseases could be prevented. However, risk assessment based on Hb analysis revealed that approximate 25% of the positive screened couples were at risk of having babies with non-severe disease, i.e. homozygous Hb E. Therefore, a simple strategy for screening out of these couples is needed to reduce unnecessary expenses of laboratory confirmation.

Development of internal quality control and proficiency testing program for thalassemia screening at Lamplaimat Hospital, Burirum province

Thalassemia screening using a combined osmotic fragility (OF) test and dichlorophenolindophenol (DCIP) precipitation test has been implemented in Lamplaimat Hospital since 2005. However, quality control (QC) system has not yet been established. In order to develop an internal QC and proficiency testing program for thalassemia screening, one of laboratory staffs was assigned to prepare QC samples by selecting left-over blood samples from routine practice. The selection criterion for positive QC was MCV \< 75 fl and Hb \> 10 g/dl. For negative QC, sample with MCV \> 85 fl and Hb \> 12 g/dl was selected. These QC samples were firstly tested using the OF and DCIP tests by the assigned staff and then blindly tested by the other two laboratory staffs who took responsibility on the OF/DCIP screening in routine practice. The screening results were recorded as unknown samples. From January to June 2009, a total of 100 QC samples (66 positive QC and 34 negative QC) were collected. All of them were sent to the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, to investigate for thalassemia and hemoglobinopathies using standard methods. Agreement of the OF/DCIP screening between the 2 staffs was evaluated using Kappa statistics.  Acceptable proficiency testing was assessed according to the criteria of no false negative OF result in a-thalassemia 1 or b-thalassemia carrier and no false negative DCIP result in Hb E carrier with Hb E \> 25% and with false positive rate of less than 20 % in normal QC samples (sample without a-thalassemia 1, b-thalassemia and Hb E). Based on Kappa analysis, a perfect agreement was obtained for both OF test (K = 0.93) and DCIP test (K = 0.96). Proficiency testing on thalassemia screening of the two staffs revealed acceptable result. The data indicates the same standard in performing thalassemia screening of the Lamplaimat’s laboratory staffs. The internal QC and proficiency testing program established in this study should prove useful for effective prevention and control of thalassemia in the region.

Screening for alpha and beta thalassemia diseases by Hb H inclusion and Hb F cell staining at Somdejprayannasangworn Hospital, Chiang Rai province

a – And b - thalassemia diseases are common in Thailand. Diagnosis of the diseases are usually made by clinical and Hb analyses. Hb H (b4) is usually detected in the Hb H disease and Hb F (a2g2) is observed in b-thalassemia. As Hb analysis is generally served at big health centers, samples from small hospitals have to be sent for analysis which usually requires at least 1 week. To provide useful data for rapid initial diagnosis at small hospital, study was done at Somdejprayannasangworn hospital, Chiang Rai province to determine the accuracy of diseases screening using Hb H inclusion and Hb F cell staining tests. Blood samples of 30 subjects who were suspected for thalassemias by the doctor and those of 32 anemic samples were initially examined for Hb H inclusion and Hb F cells at this hospital. Remaining blood samples were sent to the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, for Hb and DNA analyses. It was found that all 22 patients with Hb H disease and two a-thalassemia 1 carriers were positive for Hb H inclusion test. The sensitivity and specificity of the test for diagnosis of Hb H disease were 100 % and 95.0 %, respectively. Among 10 subjects with b-thalassemia / Hb E disease, 9 were positive for Hb F cell staining. False positives were observed in 9 subjects. The sensitivity and specificity of the test for diagnosis of b-thalassemia disease were 90.0 % and 82.7 %, respectively. These results demonstrate that Hb H inclusion and Hb F cell staining could provide useful data for initial diagnoses of a – and b-thalassemia diseases at a small hospital.

Thalassemia screening using OF/DCIP tests and turbidimetric assay

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Guideline for setecting left-over blood specimens for use as QC samples for thalassemia screening using OF/DCIP

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α/β Globin mRNA of β-thalassemia genes with premature termination

Mutation of β-globin gene causing translation-premature termination results in significant decrease of the mRNA abundance. This phenomenon arises from the degradation of mutant mRNA by nonsense codon-mediated mRNA decay (NMD). In this study, α/β-globin mRNA ratio was determined by semi-quantitative RT-PCR in β-thalassemia carriers with the β17, β41/42, β71/72 and β27 mutations and in a patient with compound heterozygous β17, β27 mutations. Values were compared with normal individual. The α/β-globin mRNA ratio of normal individual was found to be 0.98 whereas those of heterozygous for β17, β41/42, β71/72 and compound heterozygous β17/β27 were 0.89 1.66 1.60 and 3.09, respectively. Direct DNA sequencing of the cDNA demonstrated mutant mRNA only in the carrier with β17 mutation but not other mutations. This result was in concordance with the α/β-globin mRNA ratio observed. This data indicates that the α/β-globin mRNA ratio is dependent on the type and the location of premature termination mutation and related to some other factors involving NMD mechanism in cells.

Hb Bart’s and Hb E in cord blood : analysis using two automated Hb analyzers

At present, an automated hemoglobin (Hb) analyzer has been used widely for determining the Hb profiles. The aim of this study was to compare Hb Bart ‘s and Hb E levels obtained from the 2 different automated-HPLC-analyzers. One hundred and seventy-nine cord blood samples suspected of having Hb Bart’s and Hb E determined by the Primus CLC 330 (Primus Corp, MO, USA.) were recruited. These samples were analyzed again by the Variant Hemoglobin Testing System (Bio-Rad Laboratories, CA, USA.) in which the data processor was modified to quantify the amount of Hb Bart’s. All samples were investigated for α-thalassemia 1 (SEA and THAI deletions), α-thalassemia 2 (3.7 and 4.2 kb deletions), Hb Constant Spring (Hb CS) and Hb Paksé as well as Hb E genes. Analysis of the difference-values of Hb Bart’s and Hb E levels obtained from the 2 systems revealed a median (95% CI) of -0.2 (-0.3, -0.1) for Hb Bart’s and -0.15 (-0.3, -0.05) for Hb E indicating that these values were significantly different (P \< 0.001 for Hb Bart’s and P = 0.008 for Hb E; Wilcoxon sign rank test). Comparison of Hb Bart’s and Hb E levels according to the thalassemia genotypes showed a lower trend of the values obtained from the Primus in almost all genotypes. However, statistical analysis of Hb Bart’s in a group of α-thalassemia 1 newborns showed no significant difference (11.7 ± 2.0 % vs 12.1 ± 2.5 %). The results indicated that Hb Bart’s level obtained from these 2 systems might be used comparatively for screening of α-thalassemia 1 in newborns.

Problem evaluation and efficiency improvement for thalassemia and hemoglobinopathies screening at community hospitals

To improve the efficiency of thalassemia screening, problems related to the screening procedures had been assessed in 3 community hospitals i.e. Thatako Hospital; Nakornsawan province, Lansaka Hospital; Nakhon Si thammarat province, and Borabue Hospital; Mahasarakham province. The investigation was done in 3 phases. Phase I aimed to identify the problems of routine thalassemia screening. From each hospital, blood samples screened for routine services were collected and sent to the Thalassemia Research Project, the Center for Research and Development in Medical Diagnostic Laboratory (CMDL) at the Khon Kaen University for confirmation. False positive and false negative rates were determined to assess the accuracy of the results. It was found that the range of false positive and false negative rates of the osmotic fragility test (OF test) for screening of α-thalassemia 1 and β-thalassemia was 7.9-29.6% and 0-100%, respectively. The DCIP precipitation test (DCIP-test) used for Hb E screening resulted in a range for false positive results of 1.4-4.0% and for false negative results of 3.6-50%. A combination of the OF/DCIP test revealed 10.5 to 29.6% false positive- and 3.5 to 42.9%, false negative results. In phase II the causes for the measuring errors were identified. These varied from hospital to hospital. Most of the causes were related to mistakes made by the laboratory personnels. A workshop on thalassemia screening was convened to improve the skills of the laboratory staff to perform screening tests correctly. Modified laboratory procedures were implemented in each hospital. Reassessment of the efficiency of thalassemia screening was conducted again by collecting blood samples screened for thalassemia from each hospital and sent to the CMDL for confirmation. The sensitivity and specificity of the combined tests increased considerably from 57.1-96.6% to 97.1-100% and 70.4-88.9% to 81.5-89.9% indicating that the efficiency of thalassemia screening had been improved. The results indicated that the proficiency testing system should be implemented at community hospitals to standardize and improve the quality of thalassemia and hemoglobinopathies screening within Thailand.

Screening for thalassemia and hemoglobin E in pregnant Laos women at the Mother and Child Health Hospital, Vientiane, Lao People’s democratic republic

In order to provide relevant data for implementation of a prevention and control program of thalassemia in Laos population, we have studied the screening for thalassemia and hemoglobin E in pregnant Laos women. Study was conducted on 307 pregnant women attending at the Mother and Child Health hospital, Vientiane, Lao peopleûs Democratic Republic. Initial screening was performed using a combined KKU osmotic fragility (KKU-OF) and KKU dichlorophenolindophenol (KKU-DCIP) tests. Subjects were divided according to the results of OF and DCIP screening tests into 4 groups including 154 (-/-), 58 (+/-), 22 (-/+) and 73 (+/+) individuals. All blood samples were further analyzed on the Hb-HPLC analyzer and by DNA analysis by PCR to identify thalassemia genes. Among 307 subjects examined, 39 cases (12.7 %) had α-thalassemia 1, 11 cases (3.6 %) were β-thalassemia and 93 cases (30.2 %) had Hb E. The effectiveness of screening for the three severe forms of thalassemia namely; α-thalassemia 1, β-thalassemia and Hb E was calculated. The sensitivity, specificity, positive and negative predictive values were 99.2 %, 85.5 %, 83.0 % and 99.4 %, respectively. This result indicates that thalassemia screening is possible and is an effective tool for prevention and control of thalassemia and hemoglobinopathies in Lao P.D.R. With this screening approach, some false positive results might be expected but false negative for the three important forms of thalassemia should be very rare.

The association of Gγ XmnI polymorphism with hemoglobin F levels in homozygous hemoglobin E

The Gγ -Xmn I polymorphism (-158 C → T) in 131 subjects with homozygous Hb E were investigated. The Xmn I (+/+), (+/-) and (-/-) patterns were identified in 77 (58.8 %), 45 (34.3 %) and 9 (6.9 %) cases, respectively. This result indicates that majority of βE globin genes are linked to the Xmn I + allele. Among 61 cases of Hb E homozygote (Hb \> 9 g/dl) who had (bE/bE, aa/aa) genotype, 37 (59.7 %), 20 (32.3 %) and 5 (8.0 %) cases had the Xmn I (+ / +), (+ / -) and (- / -) patterns, respectively. The median levels of Hb E and other Rbc parameters in these groups were not difference. However, the median levels of Hb F as determined by Hb-HPLC analysis, the MCHF (ng) and the Hb F concentration (mg/dl) were significantly higher in the Xmn I (+ / +) group (3.6 %, 731.0 ng and 374.0 mg/dl) as compared to those with the Xmn I (+ / -) (2.4 %, 456.9 ng and 264.4 mg/dl) and the Xmn I (- / -) (2.5 %, 518.0 ng and 245.0 mg/dl) at p-value \< 0.01 using Kruskal Wallis One-way Analysis of Variance and Mann-Whitney U-Test. No difference was found between the Xmn I (+ / -) and the Xmn I (- / -) groups. Although the Gγ -Xmn I (+ / +) polymorphism is associated with high Hb F production in Hb E homozygote, it is apparent that other factors requiring further investigation might also be involved.

Effective routine screening for a - thalassemia 1 carroer with the SEA deletion

In order to provide rapid method for identifying a - thalassemia 1 in massive screening, we have tested a simple screening strategy using simple blood test and real-time PCR.\  Study was done at our ongoing thalassemia screening at the Thalassemia Service Unit, Faculty of Associated Medical Sciences, Khon Kaen University. Initial screening was done for all samples using a modified one tube osmotic fragility test (OF test) and RBC indices obtained using standard blood cell counter. Those who had positive OF test results or MCV less than 80 fl were subjected to further PCR analysis for detection of a - thalassemia 1 (SEA deletion) by two PCR methods. In the first method, a - thalassemia 1 determinant was identified using a conventional GAP-PCR routinely run in our laboratory. In the second method, identification of a - thalassemia 1 (SEA) was carried out using a real-time PCR with SyBr green I and melt curve analysis. The SEA determinant generated specific melt curve with Tm of 86 + 1 ๐ C. Among 98 subjects who were positive at the initial screening, a - thalassemia 1 (SEA deletion) was detected in 22 (22.5 %) of them by both PCR methods. These included 14 a \– thalassemia 1 carriers, 3 patients with Hb H disease and 5 subjects with double heterozygote for a-thalassemia 1 and Hb E. No a - thalassemia 1 was detected in the remaining 76 cases. This data demonstrates that identification of a - thalassemia 1 in routine practice with large number of samples can be effectively done using a combination of OF test or MCV and a real-time PCR which should prove useful in a prevention and control program of thalassemia in area with high prevalence.

Proficiency testing in laboratory diagnosis of common thalassemia and hemoglobinopathies in Thailand

To improve the efficiency of laboratory diagnosis of common thalassemia and hemoglobinopathies in Thailand,the proficiency testing program was set up at the Centre for Research and Development of Medical DiagnosticLaboratories (CMDL), Khon Kaen University. The KKU-Hb controls were sent to laboratory members togetherwith essential hematological parameters. Each time, two control samples designated as husband and wife were sent.Upon receiving, all laboratory members analyzed control samples in their routine practices and interpreted the resultusing hematological data received and result of Hb analyses in their laboratories. The result of laboratory investigationsand the interpretations as well as the risks of having fetuses with 3 severe thalassemia diseases including homozygousα- thalassemia 1, homozygous β-thalassemia and β-thalassemia / Hb E disease were applied into the form providedand sent back to CMDL. Three cycles were investigated with 21, 23 and 66 participant laboratories, respectively.All control samples were received within appropriate times and conditions. It was found that more than 90 % ofparticipant laboratories could report acceptable levels of Hb A2 and Hb F and give accurate interpretation. Memberswere analyzed and grouped into 4 different quality groups;Excellent, Good, Fair and Need improvement.The proportions of members in the Excellent, Good, Fair and Need improvement groups were respectively found tobe (81.0, 9.5, 4.75 \& 4.75 %) in the first cycle and (69.6, 0, 21.7 \& 8.7 %) in the second cycle and (56.0, 18.2,24.3 \& 1.5 %) in the third cycle. It was found that the values of Hb A2 and Hb F were reported quite accuratelyfrom each laboratory member. However, when samples with complicated data were supplied, the increased inmis-interpretation and evaluation of relative risks were observed. This result indicates the requirement of furtherimprovement in the laboratory interpretation and knowledge related to laboratory diagnosis of thalassemiaand hemoglobinopathies of the participants. The evaluation system developed should prove useful in both developmentof external quality control program in laboratory diagnosis and further facilitate the prevention and control programof thalassemia and hemoglobinopathies in Thailand.

Hb E - alpha - thalassemia at Maharaj Nakhon Si Thammarat hospital

Both hemoglobin E (Hb E) and a-thalassemia are prevalence in Thailand. Hb E-a-thalassemia syndromes are therefore common. Individuals with these syndromes are at risk for both a- and b-thalassemia diseases. In this study, the prevalence of Hb E-a-thalassemia was examined on 204 Southern Thai subjects who were encountered at the Maharaj Nakhon Si Thammarat hospital. The a-thalassemia determinants examined using PCR methods included a-thalassemia 1 (SEA type), a-thalassemia 2 (3.7 kb and 4.2 kb deletions) and Hb Constant Spring gene (aCS). Hb E gene (bE) was confirmed in all cases using allele specific PCR. The prevalence of a-thalassemia 1, a-thalassemia 2 and aCS in  this Thai  population  were found to be 2.0 %, 15.7 % and 3.9 %,  respectively. As many as 11 Hb E genotypes were observed; 71.1 % heterozygous Hb E, 1.5 % heterozygous Hb E with heterozygous a-thalassemia 1, 10.3 %  hetetozygous Hb E with  heterozygous a-thalassemia 2 (3.7 kb), 1.5 % heterozygous Hb E with  heterozygous a-thalassemia 2 (4.2 kb), 2.9 % heterozygous Hb E with heterozygous aCS, 0.5 %  heterozygous Hb E with homozygous a-thalassemia 2, 1.0 % heterozygous Hb E with compound  heterozygous a-thalassemia 2 and aCS, 6.8 % homozygous Hb E, 0.5 % homozygous Hb E with  heterozygous a-thalassemia 1, 2.4 % homozygous Hb E with  heterozygous a-thalassemia 2 and 1.5 % b-thalassemia / Hb E disease. It was found that using hematological data alone it is very difficult to differentiate all these genotypes. It is therefore recommended for those with Hb E, whose couples are carriers of a-thalassemia 1, to be examined for a-thalassemia 1 especially when the level of Hb E is less than 25 % in order to prevent the Hb Bart’s hydrops fetalis and provide appropriate genetic counseling.

Assessment of quality control producy for thalassemia screening using MCV and MCH approaches

A combined MCV/DCIP or MCH/DCIP is one of screening approach for thalassemia alternative to the OF/DCIP. Based on this approach, the MCV and MCH values obtained from electronic blood cell counters are of important that should be accurate and comparable. To evaluate the accuracy of thalassemia screening using the MCV and MCH measured from different instruments and settings, two quality control (QC) products, the RBC_A (MCV \> 80 fl and MCH \> 27 pg) and RBC_B (MCV \< 80 fl and MCH \< 27 pg), were prepared and sent to 13 participated laboratories. Rbc parameters of each QC product were measured routinely in each laboratory. By comparing to the initial values obtained in our laboratory, it was found that all Rbc parameters of the two QC products were accurately measured with the coefficient of variation (% CV) of 2.09 - 9.76 % for RBC_A and 1.11 - 11.04 % for RBC_B. Regarding to the instruments, the % CV of most Rbc parameters were less than 2 % for both RBC_A and RBC_B. To evaluate the accuracy of the MCV and MCH in application to thalassemia screening, the MCV cutoff value of 80 fl and MCH cutoff value of 27 pg were applied. The RBC_A was treated as negative sample and the RBC_B was treated as positive sample. By using the MCV approach, false negative results were obtained form 2 laboratories. In contrast, neither false negative nor false positive result was found with the MCH approach, suggesting that MCH might be more appropriate for thalassemia screening than MCV. The results indicate that these QC products might be used as an internal QC to evaluate the accuracy of Rbc parameters and as an external QC to evaluate the performance of thalassemia screening using the MCV and MCH approaches of each laboratory setting.