ORIGINAL ARTICLE Validation of the immunochromatographic strip for a-thalassemia screening: a multicenter study PRANEE WINICHAGOON, PORNNAPA KUMPAN, PAULA HOLMES, JILL FINLAYSON, CHRISTOPHER NEWBOUND, ARNOLD KABRAL, BENJAMIN LI, MANIT NUINOON, TERRY FAWCETT, CHATCHAI TAYAPIWATANA, WATCHARA KASINRERK, and SUTHAT FUCHAROEN NAKHONPATHOM, NAKHON SI THAMMARAT, AND CHIANG MAI, THAILAND; NEDLANDS, PERTH, MACQUARIE PARK, NEW SOUTH WALES, AND MELBOURNE, AUSTRALIA

a0-Thalassemia occurs from a deletion of 2 linked a-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 a0-thalassemia carriers is thus essential for the prevention and control program. An immunochromatographic (IC) strip test was developed for rapid screening of a0-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 a-thalassemia carriers with 2 a-globin genes deletion and Hb H disease. This is, in particular, the useful rapid screening test for regions where b-thalassemia and homozygous Hb E are also common. Similar hematologic and Hb data make it impossible to address the concomitant inheritance of a0-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, a-globin genotyping should be carried out in samples with positive IC strip as positive reactivity was also observed in homozygous a1-thalassemia carriers who have 2 trans a-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 a0thalassemia 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. (Translational Research 2014;-:1–7)

From the Thalassemia Research Centre, Institute of Molecular Biosciences, Mahidol University, Nakhonpathom, Thailand; Department of Hematology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, Perth, Western Australia, Australia; School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Perth, Western Australia, Australia; Department of Diagnostic Molecular Genetics, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands, Perth, Western Australia, Australia; Douglass Hanly Moir Pathology, Macquarie Park, New South Wales, Australia; School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand; Biospecifix, Melbourne, Australia; Division of Clinical Immunology, Department of Medical Technology,

Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand. Submitted for publication July 25, 2014; revision submitted October 3, 2014; accepted for publication October 29, 2014. Reprint requests: Suthat Fucharoen, Thalassemia Research Centre, Institute of Molecular Biosciences, Mahidol University, Nakhonpathom 73170, Thailand; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.10.013

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Abbreviations: CS ¼ Constant Spring; DNA ¼ deoxyribonucleic acid; Hb ¼ hemoglobin; HPLC ¼ high-performance liquid chromatography; IC ¼ immunochromatographic; MCH ¼ mean corpuscular hemoglobin; MCV ¼ mean corpuscular volume; PCR ¼ polymerase chain reaction; RBC ¼ red blood cell; Thal ¼ thalassemia

AT A GLANCE COMMENTARY Winichagoon P, et al. Background

Screening and diagnosis of a-thalassemia are essential for prevention and control of Bart’s hydrops fetalis, the most severe and lethal thalassemia disease, which is currently performed by hemoglobin (Hb) analysis, Hb H inclusion bodies, and genotyping using polymerase chain reaction technique. Translational Significance

Immunochromatographic strip test was developed for rapid screening of a-thalassemia by testing for Hb Bart’s in blood samples. In combination with hematologic data, the immunochromatographic strip test could rule out mass populations for further a0-thalassemia detection by polymerase chain reaction–based analysis, and has the potential to replace testing for Hb H inclusion bodies as it appears to be more sensitive, more specific, and less labor intensive.

INTRODUCTION

Thalassemia is an autosomal recessive inherited genetic disorder of the red blood cells (RBCs) caused by the reduction or absence of one or more globin chains of hemoglobin (Hb) molecules. a-Thalassemia is characterized by a variable degree of a-globin chain deficit. The a-globin genes (HBA1 and HBA2) are duplicated and located adjacently on the short arm of chromosome 16, where normal individuals have 4 a-globin genes. aThalassemia is most often because of a-globin gene deletion; however, molecular defects of a-thalassemias are heterogeneous.1,2 A large deletion that removes both a-globin genes (22) on 1 chromosome results in the complete abolition of a-globin production from the allele leading to a0-thalassemia, whereas a1thalassemia has the reduced a-globin gene expression because of most commonly single gene deletion that leaves 1 functional a-globin gene (2a) or to nondeletion mechanisms (aTa or aaT). The interaction between these abnormal a-globin genes

leads to many a-thalassemia syndromes. In general, the clinical severity of a-thalassemia phenotype relates to the number of affected a-globin genes.3 Individuals who have deletion or inactivation of 1 a-globin gene (a1-thalassemia) usually do not present with significant hematologic changes. When 2 a-globin genes are deleted either on the same chromosome (a0-thalassemia heterozygote) or one on each chromosome (a1thalassemia homozygote), hypochromic and microcytic RBCs are observed without anemia. Inactivation of 3 aglobin genes because of deletions with or without nondeletion a-thalassemia leads to the thalassemia intermedia, called Hb H disease, whereas the deletion of all 4 a-globin genes results in Hb Bart’s hydrops fetalis, the most severe and lethal thalassemia disease. Thalassemia is commonly found in Southeast Asia, Southern China, India, in the Middle East, and the Mediterranean region. In Thailand, the prevalence of a0thalassemia ranges between 3.6% and 10%, whereas a1-thalassemia is much more frequent, 16.4%–20%.4 Because of global migration patterns, there has been an increase in the incidence of a-thalassemia in many parts of the world including Australia and the United States of America.5 Hb Bart’s hydropic fetuses rarely survive without an intensive transfusion program and their mothers often suffer from obstetric complication.3 Screening and genetic counseling are therefore essential for the prevention and control of this severe disease, and to this end it is essential that a0-thalassemia carriers are identified. Many methods are currently used for the screening and diagnosis of thalassemia. Phenotyping by capillary electrophoresis or high-performance liquid chromatography (HPLC) and genotyping using the polymerase chain reaction (PCR) are the most precise methods for the diagnosis of thalassemia.6-11 As a small amount of Hb Bart’s is reported to appear in the RBCs of athalassemia carriers,12,13 an immunochromatographic (IC) strip test developed from monoclonal antibodies against Hb Bart’s has been used for the detection of Hb Bart’s in blood samples.14 The IC strip test is simple, easy to perform, requires no sophisticated instrumentation, and results are visualized by the naked eyes. Together with RBC indices, this immunodiagnostic kit has proven suitable for the screening and detection of a0-thalassemia carriers in large populations. In this article, we performed a multicenter study to assess

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Table I. Summary of IC strip data for a-thalassemia in samples detected by DNA analysis Thalassemia Research Center, Mahidol University (N 5 220) a-b Genotypes

Hb H and EA Bart’s 22/2a; bAbA 22/aCSa; bAbA 22/2a; bAbE 22/aCSa; bAbE 0 a -thalassemia trait 22/aa; bAbA 22/aa; bAbE Homo a1-thalassemia 2a/2a; bAbA 2a/aCSa; bAbA 2a/2a; bAbS Hetero a1thalassemia 2a/aa; bAbA 2a/aa; bAbS 2a/aa; bAbE 2a/aa; b0bE aTa/aa; bAbA Normal a-genotype aa/aa; bAbA aa/aa; b0bA aa/aa; bAbS aa/aa; bAbE aa/aa; bEbE aa/aa; b0bE aa/aa; HPFH Triplicated a-gene aa/aaa; b0bA Total

Negative

Weak

PathWest Laboratory Medicine, Nedlands, WA (N 5 89)

Positive

2

Negative

Weak

Positive

10 1 3 3

1

20 6

17

13 1 1 [25% S] 3

21 3 2

2 2 (CS)

65 10 15 4 43 1

2

166

2

8 4 [32% S]

1

1 (a1)

2 (a2)

1 (CS)

1 5

33

2

26 5 1 [41% S] 5 1

52

51

Total (N 5 309)

(N 5 18) 11 1 3 3 (N 5 45) 39 6 (N 5 15) 13 1 1 (N 5 44) 33 4 3 4 (N 5 6) (N 5 180) 93 15 1 22 5 43 1 (N 5 1) 1 309

1

a -Thal or homozygous a -thal 0

IC strip

Positive

Negative

Total

Positive Negative Total

43 1 33 5 76 2 78

11 1 5 5 16 164 1 51 5 215 231

92 217 309

Abbreviations: CS, Constant Spring; Hb, Hemoglobin; HPFH, hereditary persistence of fetal hemoglobin; IC, immunochromatographic; thal, thalassemia; WA, Western Australia. ( ), Mutated genes; [ ], % Hb S. Sensitivity 76/78 5 97.4%; specificity 215/231 5 93%. Bold indicate false positive and false negative number.

this rapid immunologic screening method for identification of a-thalassemia syndromes. MATERIALS AND METHODS Subjects. A total of 662 routinely leftover ethylenediamine tetra acetic acid blood samples from Thailand and West Australia were included in this multicenter study comprising 220 samples from the Thalassemia Research Centre at Mahidol University, Nakhonpathom, Thailand and 191 samples from Nakhon Si Thammaraj Hospital, Nakhon Si Thammaraj, Thailand. The

others were 89 samples from PathWest QEII Perth, WA, Australia and 162 samples from Douglass Hanly Moir Pathology, NSW, Australia. The project was carried out according to the principles of the Declaration of Helsinki, and study protocol was approved by the Institutional Review Board of Mahidol University. Hematologic and Hb analyses and a-globin genotyping. Hematologic data were determined by the

automatic blood cell counters being operated at individuals’ laboratory collaborated in this multicenter study. Hb typing was performed by an automated HPLC machine (VARIANT; Bio-Rad Laboratories, Hercules,

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Fig 1. Diagram shows thalassemia screening method using CBC, hemoglobin typing, and IC strip test. CBC, complete blood count; CS, Constant Spring; Hb, hemoglobin; IC, immunochromatographic; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; PCR, polymerase chain reaction; RBC, red blood cell; thal, thalassemia.

California, USA).6 PCR9-11 was used to identify a0thalassemia, a1-thalassemia, Hb H disease, and nondeletion a-thalassemia in all samples, except those from Nakhon Si Thammaraj Hospital, Thailand and Douglass Hanly Moir Pathology. In the latter, athalassemias were detected by the presence of erythrocyte Hb H inclusion bodies using the enriched young red cells from just below the buffy coat.15,16 Screening of a-thalassemia by IC strip test. To validate the modified IC strip test for screening of a-thalassemia, 100 mL of ethylenediamine tetra acetic acid blood samples were added to a 96-well plate as previously described with modification of the colloidal gold conjugate.14 Briefly the IC strip, with arrows pointing toward the sample well, is immersed vertically in the hemolyzed blood for 2–5 minutes, and the reactive bands on the test strip are interpreted visually. For positive reactivity, 2 pink bands are detected, one each at the capture line zone and the control line zone. For negative reactivity, only 1 pink band is observed in the control line zone. RESULTS Validation of IC strip test for a-thalassemia. a-Globin genotyping was carried out in 309 blood samples of various thalassemia syndromes as shown in Table I. Of these 45 samples from a-thalassemia traits were demonstrated to have a0-thalassemia gene, 18 were from patients with Hb H and EA Bart’s diseases and 15 were homozygous a1-thalassemia or compound heterozygous a1-thalassemia and nondeletion a-

thalassemia. All these samples showed strongly positive results with IC strip test except for 2 a0thalassemia samples from Thalassemia Research Centre, which had the negative results and were accounted for as false negatives. In addition, a sample of Hb S trait with homozygous a1-thalassemia (Hb S 5 25%) from PathWest QEII showed only a weakly positive result. In 44 heterozygous a1-thalassemia (2a/aa), positive reactivity was observed in 6 samples (14%) with different intensity. Among the positives, 2 samples were from patients who have also b-thalassemia/Hb E diseases (Table I). Moreover, of 6 nondeletion a-thalassemia heterozygotes, 3 heterozygous Hb Constant Spring (CS) showed positive results, and 2 nondeletion a-thalassemias with mutation on the a2-globin gene (codon 94, A / G) showed the weakly positive results, whereas 1 sample with mutation on the a1-globin gene (codon 95, G / A) showed a negative reaction. Among the 180 samples with normal a-globin genotyping, the IC strip test revealed 97.8% negative reactivity. Four positive results, 2 each of normal and heterozygous for Hb E, were identified as the falsely positive. Given the high sensitivity (97.4%) and specificity (93%) of the IC strip test for a0-thalassemia carriers and its ease of use, a rapid screening method is considered as a valuable tool. A diagram showing how to interpret the results from the IC strip test is shown in Fig 1. Evaluation of the IC strip test in a screening model for thalassemia. To evaluate the use of IC strip test for a-

thalassemia screening, 353 blood samples were taken

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Table II. Summary IC strip data for a-thalassemia in samples detected by H-body and red cell indices Nakhon Si Thammaraj (N 5 191) a-b genotypes

Hb H disease 22/2a; bAbA a0-Thalassemia trait or homo a1-thalassemia 22/aa or 2a/2a or 2a/aCSa 2 coinherited with bAbA 2 Coinherited with bAbE 2 Coinherited with bAbS Hetero a1thalassemia 2a/aa; bAbA 2a/aa; bAbS Normal a-gene aa/aa; bAbA aa/aa; b0bA aa/aa; bAbE aa/aa; b0bE aa/aa; HPFH Total

Negative

Weak

Douglass Hanly Moir Pathology, NSW (N 5 162) Positive

Negative

Weak

Positive

2

1

2

5 1 [26% S]

7

141 (8 DNA) 10 22 1 181

3 (2 DNA)

29 (3 DNA) 1 [36% S]

11 (5 DNA)

5 (DNA)

34 (7 DNA) 28 2 1 1 97

2 3

8

2

22

39 1 1

43

Total 353

(N 5 2) 2 (N 5 50)

47 1 2 (N 5 51) 50 1 (N 5 250) 182 41 24 1 2 353

a-Thal 1 or homozygous a-thal 2 IC strip

Positive

Negative

Total

Positive Negative Total

51 1 52

8 1 16 5 24 181 1 96 5 277 301

75 278 353

Abbreviations: Hb, hemoglobin; HPFH, hereditary persistence of fetal hemoglobin; IC, immunochromatographic; NSW, New South Wales; thal, thalassemia. ( ), Number of samples with DNA analysis; [ ], % Hb S. Sensitivity 51/52 5 98%; specificity 277/301 5 92%. Bold indicate false positive and false negative number.

from 2 populations, 191 Thai subjects from Southern Thailand and 162 subjects from West Australia. Hematologic data including mean corpuscular volume and mean corpuscular hemoglobin (MCV/MCH) and Hb analyses were performed, followed by the IC strip test for a0-thalassemia (Fig 1). In Douglass Hanly Moir Pathology, West Australia, a0-thalassemias were also assessed by the presence of Hb H inclusion bodies in RBCs together with the low MCV/MCH. Although phenotypes of b-thalassemia and hemoglobinopathies can be interpreted from hematologic parameters and Hb typing, it is difficult to detect heterozygous a0-thalassemia, homozygous a1-thalassemia, and heterozygous a1-thalassemia. In this cohort, positive reaction with a0-thalassemia IC strip test was found in 51 of the 52 samples that had low MCV/ MCH and positive results for erythrocyte H-inclusion bodies (Table II). These samples can be either a0-thalassemia heterozygote or homozygous a1-thalassemia and require DNA study for proper diagnosis. Two samples of Hb H disease were diagnosed by the presence of high Hb H inclusion body in erythrocytes. The absence

of the band on the capture line zone from 1 sample may be accounted for as the false negative based on the low MCV/MCH in this sample. However, weakly positive results were observed in 21 samples with normal or a1-thalassemia heterozygote and 3 samples of b-thalassemia trait (Table II). Of these 5 were proved to have normal a-globin genotype and 7 were found to be a1-thalassemia trait by PCR analysis. The remaining samples showed negative results with the IC strip test. DISCUSSION

To prevent the birth of new cases with severe a-thalassemia disease, Hb Bart’s hydrops fetalis, carrier screening, genetic counseling, and prenatal diagnosis in couples at risk are necessary. This approach may be used for population screening or screening at prenatal clinics. When the women are found to be carriers, their partners should be screened. Prevention of further thalassemic offspring in the case of couples at risk can be planned by prenatal diagnosis and selective abortion. Accurate diagnosis and understanding of gene-gene

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Fig 2. Representative IC strip test for Hb Bart’s in blood samples of a0-thalassemia heterozygote and individuals’ double heterozygous for homozygous Hb E and a0-thalassemia (lane 6) and a0b-thalassemia double heterozygote (lane 7). Hb, hemoglobin; IC, immunochromatographic.

interaction are important for genetic counseling and for the success of the prevention and control of thalassemias. Identification of a0-thalassemia carriers is the essential part for the success of this control program in many Southeast Asia countries. The laboratory diagnosis of a-thalassemia syndromes is based on the presence of Hb Bart’s, Hb H, or erythrocyte inclusion bodies. In the newborns, Hb Bart’s is present in all genotypes of a-thalassemia syndromes, and the increasing amount of Hb Bart’s is suggestive of the more severe phenotype of the diseases.17,18 In heterozygous adults for athalassemia, Hb Bart’s usually does not appear on electrophoresis or chromatography. However, small amounts of Hb Bart’s are detectable in the RBCs of athalassemia carriers.12,13 In addition, Hb H-inclusion bodies can also be detected in the RBCs, especially in the enriched young red cells population.15,16 Several methods including the RBC indices, Hb typing, and quantification of Hbs using HPLC or capillary electrophoresis and genotyping using the PCR are currently used for screening and diagnosis of a-thalassemia.6,7,9-11 PCR is the most commonly used method with reliable accuracy and precision and is becoming the gold standard method.9-11 As Hb Bart’s is present in the erythrocytes of a-thalassemia, antibodies specifically reacting to Hb Bart’s can be used to develop an immunoassay for identifying a-thalassemia carriers by an immunoprecipitation test.14,19,20 The negative IC strip test result excludes

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a0-thalassemia carrier, whereas the positive reaction will have a great benefit, especially among bthalassemia syndromes that have coinheritance of a0thalassemia (Table I). This is important in the region where a-thalassemia, b-thalassemia, and Hb E, especially homozygous Hb E, are also common as it is impossible to address the concomitant inheritance of a0-thalassemia in these samples without PCR-based techniques, resulting in misdiagnosis of individuals’ double heterozygous for a0-thalassemia and b-thalassemia or Hb E. Fig 2 shows the positive IC strip test result in these blood samples before genotyping of athalassemia by PCR technique. Although all cases with a0-thalassemia gene gave positive results, the developed IC strip test also showed positive reaction with homozygous a1-thalassemia and heterozygous Hb CS. The results are similar to those reported by Prayalaw et al.20 It is possible that there are certain amounts of Hb Bart’s in RBCs of these disorders because there are 2 a-globin gene deletions in homozygous a1-thalassemia and the Hb-CS mutation occurs on the more active a2-globin gene. In this case DNA analysis is required to confirm the presence of a0-thalassemia, as homozygous a1-thalassemia will not lead to Hb Bart’s hydrops fetalis on interaction with a0-thalassemia gene. In the previous study, the socalled false positive test result was reported with a high percentage (83%) in a1-thalassemia heterozygote.20 However, in this study, only 13.6% of blood samples from a1-thalassemia heterozygote showed the positive IC strip test result. The weakly positive result also appeared in some b-thalassemia samples, especially in b-thalassemia/Hb E, but the interpretation for b-thalassemia disease is usually based on the clinical phenotype and Hb analysis. The positive result in these samples might suggest the presence of minute amount of Hb Bart’s (g4) because of the lack of aand b-globin chains, indicating a high sensitivity of the test. Thus, the developed IC strip test in combination with PCR among the positive cases can be used for detecting and ruling out of a0-thalassemia carriers (Fig 1). These results suggest that in combination with RBC indices, the IC strip test could rule out mass populations for further a0-thalassemia detection by PCR-based analysis. The established IC strip test has an advantage because it does not require sophisticated equipments, is easy to perform, and the result can be visually interpreted without expert. It is also less-time consuming and can be done with a large number of samples. We, therefore, suggest that the IC strip test can be applied in screening program for thalassemia, which will reduce the need of expensive PCR methods in the inappropriate cases for diagnosis of a0-thalassemia carriers.

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ACKNOWLEDGMENTS

Conflicts of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and declare that Chatchai Tayapiwatana, Watchara Kasinrerk, and Suthat Fucharoen hold the patent application number 20080233659 entitled ‘‘Process of screening for alpha-thalassemia carrier using immunochromatographic strip test.’’ The assignee is the National Science and technology Development Agency (NSTDA), publication date 2008-09-25. The authors thank i 1 MED Laboratories, Thailand, for supplying the IC strips for this projects. This study was supported by Research Chair Grant, National Sciences and Technology Development Agency, Thailand (P.W. and S.F.). All authors have read the journal’s authorship agreement. The manuscript has also been reviewed and approved by all named authors. Author Contributions: P.W. contributed toward study design and data analysis. He has written and revised the manuscript. P.H., A.K., and M.N. performed research, did data interpretation, and revised the manuscript. S.F. and T.F. provided the concept and contributed toward study design and data analysis. C.T. and W.K. contributed toward study design and supplement of IC strip. P.K., J.F., B.L., and C.N. performed all experiments including DNA analysis. All authors have contributed in the preparation of final manuscript. REFERENCES

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6. Fucharoen S, Winichagoon P, Wisedpanichkij R, et al. Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC. Clin Chem 1998;44:740–8. 7. Winichagoon P, Svasti S, Munkongdee T, et al. Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system. Transl Res 2008;152:178–84. 8. Winichagoon P, Saechan V, Sripanich R, et al. Prenatal diagnosis of b-thalassaemia by reverse dot-blot hybridization. Prenat Diagn 1999;19:428–35. 9. Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common deletional determinants of a-thalassemia. Blood 2000;95:360–2. 10. Munkongdee T, Vattanaviboon P, Thummarati P, et al. Rapid diagnosis of a-thalassemia by melting curve analysis. J Mol Diagn 2010;12:354–8. 11. Pichanun D, Munkongdee T, Klamchuen S, et al. Molecular screening of the Hbs Constant Spring (codon 142, TAA.CAA, a2) and Pakse (codon 142, TAA.TAT, a2) mutations in Thailand. Hemoglobin 2010;34:582–6. 12. Wasi P, Pravatmuang P, Winichagoon P. Immunologic diagnosis of a-thalassemia traits. Hemoglobin 1979;3:21–31. 13. Makonkawkeyoon L, Ongchai S, Sanguansermsri T. Determination of hemoglobin Bart’s in a thalassemia traits by two-site radioimmunometric assay. Detection of Hb Bart’s in alpha thalassemia traits. J Med Assoc Thai 1992;75:565–9. 14. Tayapiwatana C, Kuntaruk S, Tatu T, et al. Simple method for screening of a-thalassemia 1 carriers. Int J Hematol 2009;89: 559–67. 15. Jones JA, Broszeit HK, Lecrone CN, Detter JC. An improved method for detection of red cell hemoglobin H inclusions. Am J Med Technol 1981;47:94–6. 16. Muangsapaya W, Winichagoon P, Fucharoen S, Pootrakul S, Wasi P. Improved technic for detecting intraerythrocytic inclusion bodies in a-thalassemia trait. J Med Assoc Thail 1985; 68:43–5. 17. Tanphaichitr VS, Pung-amritt P, Puchaiwatananon O, et al. Studies of hemoglobin Bart’s and deletion of a-globin genes from cord blood in Thailand. Birth Defects 1988;23(suppl 5A): 15–21. 18. Munkongdee T, Pichanun D, Butthep P, et al. Quantitative analysis of Hb Bart’s in cord blood by capillary electrophoresis system. Ann Hematol 2011;90:741–6. 19. Wanapirak C, Piyamongkol W, Sirichotiyakul S, Tayapiwatana C, Kasinrerk W, Tongsong T. Accuracy of immunochromatographic strip test in diagnosis of alpha-thalassemia-1 carrier. J Med Assoc Thail 2011;94:761–5. 20. Prayalaw P, Fucharoen G, Fucharoen S. Routine screening for alpha-thalassaemia using an immunochromatographic strip assay for haemoglobin Bart’s. J Med Screen 2014;21:120–5.

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 fe...
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