by Capillary Electrophoresis for Diagnosing β-thalassemia/ HbE Disease in Patients With Low HbF

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1 Measurement of HbA by Capillary Electrophoresis for Diagnosing β-thalassemia/ HbE Disease in Patients With Low HbF Watcharee Prasing, BSc, 1 Sakorn Pornprasert, PhD 1 * Lab Med Summer 1;5:-3 DOI: 1.139/LMGD9HES3DZRBZM ABSTRACT Objective: Capillary electrophoresis (CE) has the ability to detect hemoglobin (Hb) A in the presence of HbE. The aim of this study was to compare levels of HbA measured by CE between b-thalassemia/ HbE and homozygous HbE patients. Methods: Molecular analysis for diagnosing of b-thalassemia gene mutations and HbE was performed in blood samples containing of HbE >75% and HbF levels varying from 5% to 15% quantified by highperformance liquid chromatography (HPLC). Levels of HbA, HbE, and HbF were then measured using CE. b-thalassemia is a genetic disorder resulting in decreased or completely absent production of the normal b-globin chain usually due to nucleotide base substitution, minor deletion, or insertion. The b-thalassemia mutations at codons 17(A>T), 1/(-TCTT), 71/7(+A), and IVSI-nt1 (G>T) are commonly found in Southeast Asia. 1, Moreover, one of the most common point mutations in the β-globin gene is known as hemoglobin (Hb) E [b (B) Glu Lys, GAG>AAG], which induces alternative splicing and thus results in decreased β-globin E chain. 3-5 The compound Abbreviations Hb, hemoglobin; OF test, one-tube osmotic fragility test; RBC, red blood cell; DCIP, dichlorophenolindophenol; HPLC, high-performance liquid chromatography; LPLC, low-pressure liquid chromatography; CE, capillary electrophoresis; EDTA, ethylenediamine tetraacetic acid; MCV, mean corpuscular volume; SEA, Southeast Asian; PCR, polymerase chain reaction; HRM, high resolution melting; MARMS, multiple amplification refractory mutation system; ARMS, amplification refractory mutation system; IQR, interquartile range; IVSI, intervening sequence I 1 Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand *To Whom Correspondence should be addressed. sakornmi1@gmail.com Results: Nine patients were diagnosed with b-thalassemia/ HbE disease and 19 with homozygous HbE. All patients with b-thalassemia/hbe disease and only one patient (5%) with homozygous HbE had HbA higher than.%. Conclusions: The analysis of HbA level (>.%) by CE may be an alternative method for preliminary diagnosis of b-thalassemia/hbe with uncertain b-thalassemia/hbe disease and homozygous HbE. Keywords: b-thalassemia/hbe disease, capillary electrophoresis, HbA, HbF, homozygous HbE b-thalassemia/hbe mutations cause anemia that can vary from nearly asymptomatic to severe transfusiondependent anemia. - Due to the high cost for treatment of disease, the reduction of the number of newborns with this disease is desirable. The preliminary screening of b-thalassemia is performed in a couple by using a modified one-tube osmotic fragility test (OF test) 9 and/or red blood cell (RBC) indices 1- while the screening of HbE is performed by using dichlorophenolindophenol (DCIP) precipitation test. The confirmation test for both b-thalassemia and HbE is performed by high-performance liquid chromatography (HPLC), low-pressure liquid chromatography (LPLC), and capillary electrophoresis (CE). 1,13 Proportions of HbA /E and HbF in disease, measured by HPLC, are 3 to % and 15%, respectively, while those in homozygous HbE were 5% and <5%, respectively. 1 However, some cases of b-thalassemia/hbe disease have a high proportion of HbA /E (>75%) and a low fraction of HbF (<15%). 15 Therefore, diagnoses of both b-thalassemia/hbe disease and homozygous HbE are usually uncertain in samples that have HbA /E levels higher than 75% and HbF levels Downloaded from by guest on April 9, 1 Lab Medicine Summer 1 Volume 5, Number 3

2 varying from 5% to 15%. Molecular analysis is needed to confirm the diagnosis in these cases. However, in low-resource settings where molecular analysis is not available, HbF (a g ) levels >5% measured by HPLC, in combination with MCV <55 fl, hemoglobin <1. g/dl, and hematocrit <3% have been recommended in screening for b-thalassemia/hbe disease. 15 CE is capable of distinguishing HbA from HbE, thus permitting quantification of HbA in patients with HbE. 1 Nevertheless, whether the HbA fraction measured by CE can be diagnostic of b-thalassemia/hbe disease has not been demonstrated. Thus, the aim of this study was to compare the levels of HbA in b-thalassemia/hbe and homozygous HbE patients who have HbE levels higher than 75% and HbF levels varying from 5% to 15%. Materials and Methods Blood Samples and Thalassemia Diagnosis Blood specimens were collected in tubes containing ethylenediamine tetraacetic acid (EDTA) and submitted for investigation of thalassemia to the Associated Medical Sciences Clinical Service Center laboratory at Chiang Mai University in Chiang Mai, Thailand. Hemoglobin, hematocrit, and mean corpuscular volume (MCV) of RBCs were measured using an automated blood counter (Sysmex KX-1; Sysmex Corporation, Kobe, Japan). Quantification of HbA (for detection of b-thalassemia) and identification of hemoglobinopathies including HbE was performed using HPLC (VARIANT b-thalassemia Short Program, Bio-Rad Laboratories, Hercules, California, USA). Blood samples that had HbA /E fractions higher than 75% and HbF levels varying from 5% to 15% were analyzed by CE (CAPILLARYS, Sebia, Norcross, Georgia, USA) within hours. The α-thalassemia-1 Southeast Asian (SEA) type deletion was detected by using real-time polymerase chain reaction (PCR) with SYBR Green1 high-resolution melting (HRM) analysis as previously described. 17 DNA Preparation and Molecular Diagnosis for b-thalassemia and HbE The genomic DNA was extracted from blood samples containing HbA /E levels higher than 75% and HbF levels varying from 5% to 15% by using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). The DNA was stored at o C until analysis. The b-thalassemia codons 71/7 (+A), 1/ (-TCTT), 17 (A>T), and IVSI-nt1 (G>T) mutations were analyzed by the multiplex amplification refractory mutation system (MARMS)-PCR according to the protocol described previously. 15 The diagnosis for heterozygous and homozygous HbE was based on amplification refractory mutation system (ARMS)-PCR which has been described elsewhere. 15,1 Statistical Analysis The data are presented as median and interquartile range (IQR). Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences 11., Chicago, IL, USA). Hematological parameters and HbA, HbE, and HbF were compared between b-thalassemia/hbe disease and homozygous HbE using the Mann-Whitney U test. A P value less than.5 was considered statistically significant. Results Based on the molecular analysis, 9 patients were diagnosed with b-thalassemia/hbe disease and 19 patients with homozygous HbE. Among the 9 b-thalassemia/hbe patients, had mutations in the b-thalassemia codons 1/, had the codon 17 mutation, and 1 had the intervening sequence I (IVSI)-nt1 mutation. Moreover, of the 9 b-thalassemia/hbe disease, and 1 of the 19 patients with homozygous HbE had coinheritance of the a-thalassemia-1 SEA type deletion. The means (ranges) age of b-thalassemia/hbe disease and homozygous HbE patients were 3. (15 to 5) and.9 ( to 71) years, respectively. Median levels of MCV, hemoglobin, and hematocrit of the b-thalassemia/hbe patients were significantly lower than those of homozygous HbE patients (Figures 1A, 1B, and 1C, respectively). Whereas 1 of 19 (95%) of homozygous HbE patients had MCV higher than 55 fl, only 1 b-thalassemia/hbe patient had MCV higher than that value and this patient co-inherited the a-thalassemia-1 SEA type deletion (Figure 1A). Fourteen of 19 (7%) homozygous HbE patients and no b-thalassemia/hbe patients had hemoglobin higher than 1. g/dl (Figure 1B). Moreover, 1 of the 19 (%) homozygous HbE patients and of 9 (%) b-thalassemia/ HbE patients had hematocrit higher than.3 L/L (Figure 1C). Median HbA /E and HbF fractions determined by HPLC in b-thalassemia/hbe patients were not significantly different from those in homozygous HbE patients Downloaded from by guest on April 9, 1 Summer 1 Volume 5, Number 3 Lab Medicine 7

3 A 9 P <.1 D 1 P <.1 MCV (fl) 7 5 Hb A (%) 1 B C Hb (g/dl) P <.1 P <.1 E F Hb E (%) P =.9 P =. Downloaded from by guest on April 9, 1 Hct (L/L)..3. HbF (%) 1.1. Figure 1 Comparison in levels of MCV (A), hemoglobin (B), hematocrit (C), HbA (D), HbE (E), and HbF (F) between homozygous HbE and b-thalassemia/hbe patients. Results are shown by median (horizontal line) and IQR (whisker line). The horizontal dashed line indicates the cutoff values. The values of each sample were presented by filled cycles while those of patients with a-thalassemia-1 SEA type deletion were presented by open cycles. Lab Medicine Summer 1 Volume 5, Number 3

4 (7.% vs 3.7% and.% vs.%, P =.5 and.7, respectively). On the other hand, the hemoglobin analysis by CE showed that median levels of HbA of b-thalassemia/hbe patients were significantly higher than those of homozygous HbE patients (Figure 1D) while median levels of HbE of the former were significantly lower than those of the latter (Figure 1E). Median levels of HbF among the groups were comparable (Figure 1F). Using a HbA threshold of.% (Figure 1D), all 9 b-thalassemia/hbe patients and only 1 homozygous HbE patient, who co-inherited the a-thalassemia-1 SEA type deletion, had a HbA fraction higher than this threshold. Discussion CE has the ability to resolve and quantify HbA and HbE. 1 Thus, CE might facilitate the diagnosis of b-thalassemia/hbe disease in blood specimens that have HbE fractions higher than 75% and HbF levels varying from 5% to 15%. The present study showed that levels of HbA measured by CE in b-thalassemia/ HbE disease were significantly higher than those of homozygous HbE disease. Moreover, all b-thalassemia/ HbE patients had HbA levels higher than % while only 1 of 19 (5%) homozygous HbE patients had HbA levels higher than the cutoff. These results suggest that the occurrence of a- and d-globin chains in b-thalassemia/ HbE disease was higher than that in homozygous HbE. Only 1 homozygous HbE patient, who co-inherited the a-thalassemia-1 SEA type deletion, had a HbA level higher than %. Further, b-thalassemia/hbe patients, who co-inherited a-thalassemia-1 SEA type deletion, also had high levels of HbA (.3% and.%). This finding was consistent with a previous study that reported b-thalassemia/hbe or homozygous HbE patients who co-inherited the a-thalassemia-1 SEA type deletion had higher levels of HbA than those who did not co-inherit the a-thalassemia-1 SEA type deletion. 19 These results imply that the reduced a-globin chains in b-thalassemia/ HbE or homozygous HbE patients who co-inherited the a-thalassemia-1 SEA type deletion preferentially bind to the d-globin chain. In 1 reported study, the combination of HbF level (>5%) analyzed by HPLC with MCV (<55 fl), hemoglobin (<1. g/dl), and hematocrit (<.3 L/L) was used as an alternative method of screening for b-thalassemia/ HbE disease. 15 However, in our study, the levels of HbF measured by either HPLC or CE among b-thalassemia/hbe and homozygous HbE patients were not significantly different. Although HbE levels of homozygous HbE patients were significantly higher than those of b-thalassemia/hbe disease, their variations in both groups of patients were high. Thus, it was not possible to use the HbE levels to screening for b-thalassemia/hbe disease and homozygous HbE. All b-thalassemia/hbe patients had hemoglobin levels lower than 1. g/dl; however 5 of 19 (%) homozygous HbE patients also had hemoglobin levels lower than this threshold. In addition, of 9 (%) b-thalassemia/ HbE patients had hematocrits higher than.3 L/L. Therefore, it is also difficult to use hemoglobin and hematocrit to classifying b-thalassemia/hbe disease and homozygous HbE. Most of the b-thalassemia/hbe patients (9%) had MCV lower than 55 fl; however, there was a case of homozygous HbE which had MCV lower than this value. Moreover, among b-thalassemia/hbe patients who co-inherited the a-thalassemia-1 SEA type deletion, 1 patient had MCV higher than 55 fl while the other patient had MCV less than 55 fl. This observation was consistent with a previous study that reported MCV levels of homozygous HbE patients with and without co-inheritance of a-thalassemia-1 SEA type deletion varied from 51 1 fl and 7 fl, respectively. Thus, the use of MCV only is inadequate for screening of b-thalassemia/hbe disease and homozygous HbE. Increased HbA is specific for diagnosis of b-thalassemia; thus, the combination of HbA level and MCV might be helpful to differentiate between b-thalassemia/hbe disease and homozygous HbE. In conclusion, b-thalassemia/hbe disease may be differentiated from homozygous HbE in specimens that have HbA /E fractions higher than 75% and HbF fractions varying from 5% to 15% by using a combination of the HbA level >% measured by CE, and MCV <55 fl. This approach might be used to decrease the number of specimens referred for molecular testing, which would be especially benefician in low-resource settings where b-thalassemia and HbE are prevalent. LM Acknowledgments The authors thank the technicians at the Associated Medical Sciences Clinical Service Center, Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand for their help and assistance. We are also grateful to Roscoe C. Butler, Jr., for editing the manuscript. Downloaded from by guest on April 9, 1 Summer 1 Volume 5, Number 3 Lab Medicine 9

5 References 1. Chamras U, Sukunthamala K, Pornprasert S. SYTO9 and SYBR GREEN1 with high resolution melting analysis for molecular confirmatory testing of the common Southeast Asian beta()- thalassemia mutations. Hemoglobin. 9;33: Mirasena S, Shimbhu D, Sanguansermsri M, Sanguansermsri J. Detection of beta-thalassemia mutations using a multiplex amplification refractory mutation system assay. Hemoglobin. ;3: Fucharoen S, Winichagoon P. Thalassemia in SouthEast Asia: problems and strategy for prevention and control. Southeast Asian J Trop Med Public Health. 199;3: Fucharoen S and Winichagoon P. Hemoglobinopathies in Southeast Asia: molecular biology and clinical medicine. Hemoglobin. 1997;1: Traeger J, Wood WG, Clegg JB, Weatherall DJ. Defective synthesis of HbE is due to reduced levels of beta E mrna. Nature. 19;: Fucharoen S, Ketvichit P, Pootrakul P, Siritanaratkul N, Piankijagum A, Wasi P. Clinical manifestation of beta-thalassemia/hemoglobin E disease. J Pediatr Hematol Oncol. ;: Fucharoen S, Winichagoon P, Pootrakul P, Piankijagum A, Wasi P. Variable severity of Southeast Asian beta -thalassemia/hb E disease. Birth Defects Orig Artic Ser. 197;3:1-.. Winichagoon P, Fucharoen S, Chen P, Wasi P. Genetic factors affecting clinical severity in beta-thalassemia syndromes. J Pediatr Hematol Oncol. ;: Sirichotiyakul S, Tantipalakorn C, Sanguansermsri T, Wanapirak C, Tongsong T. Erythrocyte osmotic fragility test for screening of alpha-thalassemia-1 and beta-thalassemia trait in pregnancy. Int J Gynaecol Obstet. ;: Nathalang O, Nillakupt K, Arnutti P, Boonsiri T, Panichkul S, Areekul W. Screening for thalassemia and hemoglobinopathy in a rural area of Thailand: a preliminary study. J Med Assoc Thai. 5;(S3): Tatsumi N, Tsuda I, Funahara Y, et al. Automatic measurement of hemoglobin F in blood obtained from patients with hemoglobin E/E and beta-thalassemia/hemoglobin/e. Southeast Asian J Trop Med Public Health. 199;3(S): Tatsumi N, Tsuda I, Funahara Y, Bunyaratvej A, Pootrakul A, Fucharoen S. Analysis of hematological data of thalassemia cases in Thailand. Nihon Ketsueki Gakkai Zasshi. 199;5: Van Delft P, Lenters E, Bakker-Verweij M, et al. Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations. Int J Lab Hematol. 9;31: Pornprasert S, Kasemrad C, Sukunthamala K. Diagnosis of thalassemia on dried blood spot samples by high performance liquid chromatography. Hemoglobin. 1;3: Pornprasert S, Moriyama A, Kongthai K, et al. Detection of betathalassemia/hemoglobin E disease in samples which initially were diagnosed as homozygous hemoglobin E. Clin Lab. 13;59: Mais DD, Gulbranson RD, Keren DF. The range of hemoglobin A() in hemoglobin E heterozygotes as determined by capillary electrophoresis. Am J Clin Pathol. 9;13: Pornprasert S, Phusua A, Suanta S, Saetung R, Sanguansermsri T. Detection of alpha-thalassemia-1 Southeast Asian type using real-time gap-pcr with SYBR Green1 and high resolution melting analysis. Eur J Haematol. ;: Old J, Traeger-Synodinos J, Galanello R, et al. Molecular diagnosis: Prevention of Thalassemias and other Hemoglobin Disorders. Nicosia Team up Creations Ltd, Nicosia: Thalassemia International Federation; 5: Sae-ung N, Srivorakun H, Fucharoen G, Yamsri S, Sanchaisuriya K, Fucharoen S. Phenotypic expression of hemoglobins A, E and F in various hemoglobin E related disorders. Blood Cells Mol Dis. ;: Pornprasert S, Treesuwan K, Punyamung M, Kongthai K. Hb A/E levels found in co-inheritance with the α-thalassemia-1--(sea)/ type deletion and either Hb E or β-thalassemia. Hemoglobin. ;3: To read this article online, scan the QR code, ascpjournals.org/content/5/3/. full.pdf+html Downloaded from by guest on April 9, 1 3 Lab Medicine Summer 1 Volume 5, Number 3