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.

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.

Common abnormal hemoglobins found in Thailand and laboratory diagnostics

Since the introduction of the national prevention and control program of thalassemia and hemoglobinopathies throughout Thailand, number of request for hemoglobin analysis have been dramatically increased. Numbers of dedicated hemoglobin analyzers including low pressure liquid chromatography (LPLC), high pressure liquid chromatography (HPLC) and capillary electrophoresis (CE) have been used by many diagnostic laboratories. This has led to the observations of many abnormal hemoglobins in Thailand, most of them could not be accurately diagnosed at routine setting. Further study at the molecular level has provided useful information related to the molecular basis and the development of molecular diagnostics for many of these hemoglobin variants. In this article, common hemoglobin variants encountered at the Centre for Research and Development of Medical Diagnostic Laboratories, Khon Kaen University, one of the referral centers for thalassemia and hemoglobinopathies, were summarized. They were grouped according to the frequencies observed, types of globin gene defects and analytical characteristics observed at routine which are useful for the initial prediction of abnormal hemoglobins and selection of appropriate molecular diagnostics for final diagnosis.

Effects of heparin on thalassemia genes analysis by PCR

EDTA-anticoagulated blood is usually recommended for use in routine analysis of thalassemia genes. However, collection of blood from the fetus or newborns is usually done using heparin because of its more potent anti-coagulant activity. However, since heparin can inhibit Taq polymerase enzyme used in PCR, the use of heparinized blood for PCR analysis may be affected. ACD, another blood anti-coagulant with a preservative property may be used as an alternative anti-coagulant. In this study, the effects of EDTA, heparin and ACD anti-coagulants on PCR analysis of a – thalassemia 1 (SEA deletion) and bE – globin gene were compared. a – thalassemia 1 was detected using gap – PCR and bE – globin gene was identified by allele specific PCR assay. It was found that both EDTA and ACD anti-coagulants had no effect on the efficiency of PCR analysis of the two thalassemia genes. However, substantial reduction in the efficiency of PCR analysis was observed with the use of heparin as an anti-coagulant at a concentration over 25 IU/ml. Amplification efficiency was improved when heparinized blood DNA was diluted prior to PCR analysis. The result from this study should prove useful for development of a guideline of blood collection for routine PCR analysis of halassemia.                              

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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Implementation of quality control system for improvement of thalassemia screening at Maung Saung hospital, Roiet province

The prevention and control program of thalassemia and hemoglobinopathies has been carried out at Maung Saung Hospital, Roiet province according to the policy of the Ministry of Public Health. Discrepancy between the result of thalassemia screening and confirmatory test is commonly encountered. This study aimed to implement the quality control system at the hospital for improvement of thalassemia and hemoglobinopathies screening using a combined osmotic fragility test (KKU-OF) and dichlorophenolindophenol test (KKU-DCIP-Clear). Study plan was designed and made into 3 phases. In Phase I, a retrospective data in the year 2006 was collected and analyzed to identify to the screening problems. There were 5.4 % false positive and 5.4 % false negative for DCIP test but the effectiveness of OF test could not be determined as α - thalassemia 1 has not been examined. Phase II was an implementation of quality control system for thalassemia screening. Staffs performing screening tests were re-trained at the Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Khon Kaen University. Standard operating procedures (SOP) for KKU-OF and KKU-DCIP-Clear were then prepared. Phase III was set to evaluate the effectiveness of screening after implementation of the system. Prospective screening was carried out at Maung Saung hospital on 130 subjects and the remaining blood specimens were sent to the CMDL for further confirmatory tests. Among 130 subjects examined,α - thalassemia 1, β - thalassemia and Hb E were identified in 3.9 %, 0.8% and 40.0 %, respectively. The sensitivity, specificity, positive predictive and negative predictive values were found to be 100 %, 95.5 %, 95.0 % and 100 %, respectively. This result indicates that thalassemia and hemoglobinopathies screening at Maung Saung Hospital has been improved which should consequently lead to a more effective prevention and control program.

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.

Eight years of the Master of Science Program in Medical Science, Faculty of Associated Medical Sciences, Khon Kaen University

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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 screening for a-thalassemia 1, b-thalassemia and Hb E at the Diagnostic Clinical Microscopy Unit, Srinagarind Hospital

According to the policy of  the Ministry of  Public Health, severe thalassemia diseases targeted for prevention and control include homozygous a - thalassemia 1 (Hb Bart’s hydrops fetalis), homozygous b - thalassemia and b - thalassemia / Hb E disease. The objective of this study is to evaluate the effectiveness of thalassemia screening using a combined MCV (cut off; 80 fl) and KKU-DCIP-Clear at the Diagnostic Clinical Microscopy Unit, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University. Studied was carried out on 100 subjects. After screening, definite diagnosis of each case was done by standard Hb and DNA analyses. Thalassemia identified included 1 b - thalassemia trait, 1 a - thalassemia 1 trait, 1 Hb E trait with a - thalassemia 1 trait, 5 Hb E trait with a - thalassemia 2, 2 Hb E trait with Hb Con Sp trait, 1 Hb E trait with a - thalassemia 2 / Hb Con Sp, 24 Hb E trait, 3 Homozygous Hb E, 1 homozygous Hb E with Hb Pakse’ trait, 12 a - thalassemia 2 trait, 1 a - thalassemia 2 / Hb Con Sp , 1 Hb Lepore trait and 47 non-thalassemia. No false negative was found for screening of the three important thalassemia carriers but 6 false positive cases were encountered. Therefore, the sensitivity and specificity  of screening for a - thalassemia 1, b - thalassemia and Hb E using MCV and KKU-DCIP-Clear were 100 % and 90.2 %, respectively. The specificity was increased to 91.8 % when another red blood cell parameter, mean cell hemoglobin (MCH) (cut off; 27 pg) was used additionally. This study confirmed that the MCV and MCH values obtained from quality controlled electronic blood cell counter could be used for screening of a and b - thalassemias instead of the OF test. Using MCV and MCH in combination with DCIP test should provide effective screening for severe thalassemia carriers in routine practice.      

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.

Thalassemia screening in Thailand

The objective of thalassemia screening in Thailand is to identify a - thalassemia 1, b - thalassemia and Hb E carriers. Those with these thalassemia genes will be given appropriate genetic counseling in a prevention and control program. The aim is to prevent birth of new case with three severe thalassemia diseases including homozygous a - thalassemia 1, homozygous b - thalassemia and b - thalassemia / Hb E disease. In general, two screening strategies are performed. In the first approach, a and b - thalassemias are screened for using a one tube osmotic fragility test (OF-test) and Hb E was identified by DCIP precipitation test (DCIP test). Alternatively, screening can be done using a combined RBC indices (mean cell hemoglobin; MCH and/or mean cell volume; MCV) and a DCIP test. While positive sample at this preliminary screening will be investigated further by Hb and DNA analyses, no further test is required for those with negative result. Both screening strategies can be done effectively but application depends on laboratory facilities and technical skill of the personnel. However, quality of thalassemia screening in routine practice needs to be evaluated regularly in order to minimize false negative result.

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.

Interpretation of hemoglobin analysis for diagnosis of common thalassemia in Thailand

At present hemoglobin (Hb) analysis is mainly performed using automated hemoglobin analyzer based on eitherhigh pressure liquid chromatography (HPLC), low pressure liquid chromatography (LPLC) or capillary zoneelectrophoresis (CZE). These automated Hb analyzers separate each Hb and quantify its amount by builtin spectrophotometric determination. Hb chromatogram or electrophoregram as well as the amount of each Hbare shown and could be printed. Interpretation of the result of Hb analysis is usually made by investigator whoperforms the test. Accurate interpretation and diagnosis require basic knowledge on both the principle ofthe machine used and the heterogeneity of thalassemia which is the most common inherited genetic disorderin Thailand. Using subject information including age, clinically signs and symptoms, history of blood transfusionand the result of initial screening test in this interpretation and diagnosis should help in providing more accurateinterpretation and diagnosis of common forms of thalassemia.

A visit to the Medical Technology students at Somdej Praborom Rajchathewi hospital

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