Abstract
Primary screening for thalassemia carriers usually involves an accurate blood count using an automated blood cell analyzer. We analyzed the red cell and reticulocyte parameters from 200 samples of various types of thalassemias and identified the discrimination criteria for differential diagnosis. These were separated into four groups based on genotypes. These groups included α-thalassemia, β-thalassemia, β-thalassemia with Hb E and Hb E trait, which are the important target of thalassemia screening. To compare the effectiveness of the screening strategies, seven selected screening tools were compared, including MCV alone (cutoff <80 fL); MRV alone (cutoff <100 fL); SD-C-NR alone (cutoff >28.5); a combined MCV and MRV; a combined MCV and SD-C-NR; a combined MRV and SD-C-NR; and a combined MCV, MRV and SD-C-NR. The combination of MCV, MRV and SD-C-NR has highest sensitivity for discrimination in all thalassemia, β-thalassemia, α-thalassemia, Homozygous Hb E and Hb E trait groups as 99.5, 100, 98, 100 and 100 %, respectively. The effectiveness of the combination of MCV, MRV and SD-C-NR parameters in the present study was determined as the faster and higher sensitivity screening tool than the other methods, including simple, reliable, cost-effective, and using only one automated hematology analyzer, saving labor costs.
Keywords: Thalassemia syndrome carrier, Red blood cell indices, Reticulocyte parameters, Automated blood cell analyzer
Introduction
Thalassemia syndrome is the most common genetic disorder of microcytic and hemolytic anemias, about 250 million people in the world carry thalassemia genes mainly in the Mediterranean area, the Middle East, the Indian subcontinent, and in Southeast Asia [1]. This genetic disorder results in defective globin synthesis, poorly or absent synthesized of α-globin chains in α-thalassemia and β-globin chains in β-thalassemia. The gene–gene interactions in this population can lead to cause several types of thalassemia syndrome. The three prime targets of prevention and control are homozygous α0-thalassemia (causing the hemoglobin (Hb) Bart hydrops fetalis), homozygous β-thalassemia, and β-thalassemia/Hb E [2]. This imbalanced synthesis results in a variable degree of anemia which stimulates erythropoietin production, leading to proliferation and expansion of the bone marrow. The β-thalassemias have a considerable phenotypic variation depending on multiple factors, which include the nature of the mutation involved. This leads to a wide range of presentations from profound anemia (requiring lifelong blood transfusions—β-thalassemia major) to extremely mild anemia (β-thalassemia trait) [3].
In Thailand, about 1 % of Thai people (600,000 persons) were thalassemia patients [4] and about 40 % of Thai people have a thalassemia trait [5]. Therefore, in the prevention and control program are concern for the rapid, accurate, and inexpensive screening protocols to identify carriers of α0-thalassemia, β-thalassemia, and Hb E, especially in a prenatal population at risk for Hb disorders.
The conventional method for primary screening of all forms of thalassemia relies on hematologic index cutoffs analyzed by an accurate blood cell count analyzer. Mean corpuscular volume (MCV) values less than 80 femtoliter (fL) and mean corpuscular hemoglobin (MCH) values less than 27 picogram (pg) are examined as cutoff to confirm or exclude the diagnoses of α-thalassemia and β-thalassemia [6, 7]. However, it has been demonstrated that a single-tube osmotic fragility (OF) test, it might be an attractive alternative to identify carriers of α- and β-thalassemias [8–10]. The used of a combined modified OF test and modified dichlorophenolindophenol (DCIP) test for Hb E [11] has been proposed for screening in rural communities of Southeast Asia [12].
Reticulocytes are juvenile red blood cells. They contain the ribosomal ribonucleic acid remnants which appear in large amounts in the cytoplasm of the nucleated precursor red blood cells. The number of reticulocytes in the peripheral blood is an accurate reflection of erythropoietic activity assuming that reticulocytes are released from the bone marrow after the normal time, and that they remain in circulation for the normal period of time [13]. Now-a-days all automated blood cell analyzers, which were developed under the advance technologies, can be provide erythrocyte and reticulocyte parameters for screening these various types of thalassemias discrimination from the normal individuals. The aim of the present study was to analyze the red cell indices and reticulocyte parameters provided by the Coulter LH 750 automated analyzer for discrimination of thalassemia carriers.
Materials and Methods
Subjects
Study participants included 300 apparently healthy couples, pregnant women and anyone who suspected the hemoglobin disorders. Sample selection was those who consecutively attended for care service between January and December 2014 at Thalassemia clinic at Sawanpracharak Hospital, Nakhonsawan Province, Thailand. Our study was approved by the institutional ethical committee of Naresuan University, Phitsanulok, Thailand. After informed consent was obtained EDTA-anticoagulated blood samples were obtained and transferred on ice within 2 h to the Medical Technology Laboratory Unit of Sawanpracharak Hospital where all laboratory testing was performed.
Screening and Hematologic Analysis
Complete Blood Counts (CBC) and RBC indices were determined using the Coulter LH 750 automated analyzer (Beckman Coulter, Fullerton, CA USA).
Reticulocyte Analysis
Reticulocyte indices were also determined using the Beckman Coulter LH 750 analyzer. The procedure of reticulocyte analysis in this analyzer uses new methylene blue dye (supravital dye) incubation with whole-blood samples, to precipitate the basophilic RNA network within the reticulocytes. The function of the reticulocyte stain is to identify and delineate the reticulocytes from mature red cells. Hemoglobin and unbound stain are removed by using a clearing reagent, then leaving clear spherical mature reticulocytes and darkly stained reticulocytes. Stained reticulocytes are differentiated from mature red cells and other cell populations by light scatter, direct current measurements, and opacity characteristics. Once the cells have been stained and cleared, the volume, conductivity and light scatter (VCS) Technology of reticulocyte analysis is employed, utilizing a unique flow cytometric means of cell interrogation to count and classify reticulocytes. All reticulocytes are identified and classified by simultaneous three-dimensional analysis using Volume, Conductivity, and Light Scatter.
Hemoglobin Analysis
Beta-Thalassemia traits were examined in samples with Hb A2 levels exceeding 4.0 % using high performance liquid chromatography in our laboratory (HPLC) [18, 19]. Hb Constant Spring (Hb CS) was also examined by using HPLC. We also confirmed the separation and quantitation of Hb A2 and Hb E by using automated capillaries zone electrophoresis (Sebia, Bio-Rad) [14]. The capillaries system uses the principle of capillary electrophoresis in free solution. Electrophoresis was performed in alkaline buffer (pH 9.4) allowing separation to be directed by pH and endosmosis. All Hb-types concentrations were measured at a wavelength of 415 nm.
DNA Analysis
The polymerase chain reaction (PCR) analysis of genomic DNA extraction from peripheral blood leukocytes was used as gold standards for α-thalassemia. Alpha-thalassemia0 mutations (SEA type) were identified by allele-specific PCR [15–17].
Statistical Analysis
Descriptive statistics, median and interquatile range in non-parametric data were used to describe hematologic features of all subjects. All effectiveness variables from Coulter LH 750 auto-analyzer based on RBC and reticulocyte indices in each group were compared by using Mann-Whiteney U-test. We combined the red cell and reticulocyte parameters for thalassemias and Hb E screening. We also calculated the sensitivity and specificity of the red cell and reticulocyte parameters by using Receiver Operating Characteristic (ROC) analysis. All analysis was performed by SPSS version 13.0 (SPSS, Chicago, IL, USA).
Results
The results of the thalassemia types are summarized in Table 1. Among 300 participants studied, 200 (66.7 %) were found to carry thalassemias or hemoglobinopathies. No thalassemia was detected in the remaining 100 subjects (33.3 %). The results and comparison of these groups are demonstrated in Table 2. The comparison results of all hematologic characteristics of each group of thalassemia participants are demonstrated in Table 3. As expected, the most common genotype was Hb E heterozygote, which was identified in 100 subjects, included 12 β-thalassemia/Hb E, 40 homozygous Hb E and 48 Hb E trait. In the former group, fifty β-thalassemia traits were detected. Fifty α-Thalassemias, including 18 α0- thalassemia traits (SEA type), 14 Hb H, 7 Hb H/CS and 11 Hb CS traits were identified and summarized in Table 1.
Table 1.
Thalassemia genotypes observed in 200 carriers
| Thalassemia genotype | No. of cases |
|---|---|
| α-Thalassemia | 50 |
| α0-Thalassemia trait (SEA type) | 18 |
| Hb H | 14 |
| Hb H/CS | 7 |
| Hb CS trait | 11 |
| β-Thalasemia | 50 |
| β-Thalasemia trait | 50 |
| β-Thalassemia with Hb E/Homozygous Hb E | 52 |
| Homozygous Hb E | 40 |
| β-Thalasemia/Hb E | 12 |
| Hb E trait | 48 |
Table 2.
Comparison of all blood cell indices of normal control and β-thalassemia, α-thalassemia and hemoglobin E carrier
| Variables | Normal (n = 100) | All thalassemia carrier (n = 200) | p value |
|---|---|---|---|
| Age (years) | 35.9 (8.5–44.0)* | 23.10 (2.85–41.90)* | 0.057 |
| WBC (×109/L) | 7.30 (5.63–8.60) | 8.50 (6.83–10.30) | 0.001 |
| RBC (×1012/L) | 3.685 (3.263–4.175) | 4.620 (3.538–5.378) | <0.001 |
| Hb (g/dL) | 10.65 (9.03–12.10) | 9.20 (7.70–10.90) | <0.001 |
| Hct (%) | 31.7 (27.4–36.2) | 29.45 (25.23–34.28) | 0.019 |
| MCV (fL) | 88.40 (83.88–91.90) | 66.55 (60.63–72.55) | <0.001 |
| MCH (pg) | 29.25 (27.53–30.80) | 20.70 (18.93–22.48) | <0.001 |
| MCHC (g/dL) | 33.15 (32.50–33.80) | 31.40 (30.50–32.10) | <0.001 |
| RDW (%) | 14.60 (13.80–17.10) | 19.55 (17.10–26.55) | <0.001 |
| Plt (×109/L) | 248.50 (195.50–345.25) | 253.00 (198.00–347.75) | 0.783 |
| % Ret count (%) | 1.80 (1.29–2.63) | 2.41 (1.58–3.09) | 0.001 |
| Ab Ret (×106/L) | 0.0649 (0.0476–0.0904) | 0.1046 (0.0674–0.1445) | <0.001 |
| MRV (fL) | 110.05 (102.48–116.35) | 95.00 (86.25–99.98) | <0.001 |
| IRF (ratio) | 0.370 (0.310–0.420) | 0.295 (0.200–0.378) | <0.001 |
| M-V-Ret | 54.70 (51.25–57.75) | 47.40 (43.00–50.10) | <0.001 |
| M-C-Ret | 73.25 (69.90–77.80) | 87.05 (82.80–93.18) | <0.001 |
| M-S-Ret | 137.85 (128.80–144.53) | 140.35 (127.80–151.10) | 0.091 |
| SD-V-Ret | 14.40 (12.95–16.02) | 16.63 (14.69–18.76) | <0.001 |
| SD-C-Ret | 25.32 (23.02–27.35) | 30.79 (27.55–34.51) | <0.001 |
| SD-S-Ret | 18.19 (16.95–19.96) | 16.96 (15.25–19.15) | <0.001 |
| M-V-NR | 46.40 (44.03–48.48) | 35.40 (33.13–38.68) | <0.001 |
| M-C-NR | 72.70 (69.95–77.70) | 90.00 (85.05–96.88) | <0.001 |
| M-S-NR | 78.30 (65.78–84.60) | 78.25 (61.83–87.35) | 0.454 |
| SD-V-NR | 10.91 (10.41–11.50) | 11.29 (10.00–13.35) | 0.140 |
| SD-C-NR | 24.73 (22.88–26.26) | 30.58 (30.23–33.17) | <0.001 |
| SD-S-NR | 14.04 (13.15–15.43) | 16.76 (13.95–20.24) | <0.001 |
* Median and interquartile range
Table 3.
Comparison of all blood cell indices of α-thalassemia, β-thalassemia and hemoglobin E carrier
| Variables | α-Thalasemia (n = 50) | β-Thalasemia (n = 50) | β-Thal with Hb E/Homo Hb E (n = 52) | Hb E trait (n = 48) | p value |
|---|---|---|---|---|---|
| Age (years) | 24.5 (2.1–50.9)* | 18.30 (1.0–40.9)* | 33.5 (1.2–62.9)* | 33.3 (3.1–58.9)* | 0.09 |
| WBC (×109/L) | 8.7 (6.4–10.50) | 8.2 (6.9–9.9) | 8.7 (6.9–10.2) | 8.1 (6.8–9.8) | 0.719 |
| RBC (×1012/L) | 4.71 (3.46–5.44) | 4.61 (3.97–5.71) | 4.30 (3.26–5.130) | 4.18 (3.78–4.80) | 0.263 |
| Hb (g/dL) | 9.2 (7.5–11.2) | 9.69 (8.0–11.1) | 9.2 (7.6–10.3) | 9.4 (8.0–10.9) | 0.580 |
| Hct (%) | 29.2 (25.0–35.5) | 31.9 (26.0–37.6) | 28.7 (24.1–32.9) | 29.8 (25.5–33.5) | 0.160 |
| MCV (fL) | 66.1 (60.4–70.4) | 70.7 (62.9–78.90 | 65.2 (60.3–69.60 | 71.2 (66.1–75.5) | 0.003 |
| MCH (pg) | 20.7 (19.2–22.30 | 21.0 (18.3–23.0) | 20.4 (19.2–22.7) | 22.9 (20.2–24.3) | 0.019 |
| MCHC (g/dL) | 31.7 (31.1–32.1) | 30.5 (27.78–32.10) | 31.6 (30.8–32.2) | 31.9 (30.8–32.7) | 0.001 |
| RDW (%) | 18.4 (16.5–27.8) | 21.55 (15.68–26.10) | 19.4 (17.7–26.70) | 17.1 (15.2–21.4) | 0.015 |
| % Ret count (%) | 2.40 (1.38–2.97) | 2.68 (1.83–4.05) | 2.25 (1.55–2.85) | 1.52 (1.11–2.13) | <0.001 |
| Ab Ret (×106/µL) | 0.098 (0.065–0.141) | 0.117 (0.085–0.1660) | 0.103 (0.062–0.139) | 0.066 (0.046–0.085) | <0.001 |
| MRV (fL) | 93.80 (85.35–102.60) | 98.65 (86.00–107.88) | 94.85 (86.78–98.00) | 91.3 (87.28–96.95) | 0.061 |
| IRF (ratio) | 0.28 (0.22–0.350) | 0.35 (0.23–0.403) | 0.27 (0.19–0.380) | 0.28 (0.23–0.34) | 0.162 |
| M-V-Ret | 46.309 (41.48–50.55) | 49.35 (42.98–53.830) | 47.40 (43.38–49.28) | 47.30 (44.15–49.08) | 0.392 |
| M-C-Ret | 89.85 (84.23–95.150 | 84.95 (78.43–91.30) | 86.90 (84.53–92.03) | 81.75 (74.85–88.3) | <0.001 |
| M-S-Ret | 141.55 (128.18–151.43) | 143.75 (129.30–154.13) | 136.70 (121.20–149.23) | 115.20 (109.73–126.13) | <0.001 |
| SD-V-Ret | 16.13 (14.60–18.49) | 16.36 (14.17–18.200) | 17.29 (15.23–20.01) | 15.23 (12.84–18.00) | 0.002 |
| SD-C-Ret | 31.23 (27.54–34.29) | 30.95 (27.14–34.84) | 30.53 (27.62–34.77) | 27.79 (24.66–31.67) | 0.019 |
| SD-S-Ret | 16.52 (15.23–17.62) | 15.93 (13.88–19.29) | 17.64 (16.53–20.64) | 17.77 (15.72–20.10) | 0.002 |
| M-V-NR | 35.00 (33.25–38.88) | 36.10 (32.63–40.15) | 35.25 (33.25–37.38) | 38.45 (36.23–40.10) | 0.010 |
| M-C-NR | 91.70 (83.78–100.30) | 88.45 (84.00–95.45) | 90.00 (87.83–95.98) | 80.10 (75.18–86.03) | <0.001 |
| M-S-NR | 81.60 (73.05–88.18) | 81.40 (65.60–94.40) | 69.50 (57.88–80.23) | 54.10 (49.15–63.30) | <0.001 |
| SD-V-NR | 10.90 (9.47–13.32) | 11.65 (9.76–13.57) | 11.59 (10.29–12.88) | 10.09 (9.56–11.14) | 0.001 |
| SD-C-NR | 31.77 (28.95–35.80) | 31.47 (27.31–34.02) | 30.58 (30.58–30.58) | 30.58 (30.58–30.58) | 0.019 |
| SD-S-NR | 15.33 (13.23–19.27) | 17.65 (14.08–21.06) | 17.15 (15.24–20.43) | 13.95 (12.74–15.73) | <0.001 |
* Median and interquartile range
We used the ROC curve to calculate the area under curve (AUC) of the important of red cell and reticulocyte parameters. We found that MCV, MCH, Mean reticulocyte volume (MRV) and Standard deviation conductivity non-reticulocyte (SD-C-NR) are the important parameters for this discrimination and the others as demonstrated in Fig. 1. To compare the effectiveness of the screening strategies, the data were collected as shown in Table 3 were separated into 4 groups based on genotypes. These groups included α-thalassemia, β-thalassemia, β-thalassemia with Hb E/Homozygous Hb E and Hb E trait, which are the important targets of thalassemia screening. Seven selected screening tools were compared, including MCV alone (cutoff <80 fL); MRV alone (cutoff <100 fL); SD-C-NR alone (cutoff >28.5); a combined MCV and MRV; a combined MCV and SD-C-NR; a combined MRV and SD-C-NR; and a combined MCV, MRV and SD-C-NR. As shown in Table 4, the combination of MCV, MRV and SD-C-NR has highest sensitivity for discrimination in all thalassemia, α-thalassemia, β-thalassemia, β-thalassemia with Hb E/Homozygous Hb E and Hb E trait groups as 99.5, 100, 98, 100 and 100 %, respectively. Based on these results, the effectiveness of the combination of MCV, MRV and SD-C-NR parameters in the present study was determined as the faster and higher sensitivity screening tool than the other methods.
Fig. 1.
Demonstration of the AUC of each parameter was used for the thalassemia carrier screening
Table 4.
Demonstration of the effectiveness of Thalassemia and hemoglobinopathy screening among 200 carriers using MCV and the combination of MRV and SD-C-NR
| Parameters | Sensitivity (%) | Specificity (%) | ||||
|---|---|---|---|---|---|---|
| All Thallassemia | α-Thalasemia | β-Thalasemia | β-Thal with Hb E/Homo Hb E | Hb E trait | ||
| MCV <80 fLor | 94.0 | 94.0 | 88.0 | 98.1 | 94.0 | 92.0 |
| MRV <100 | 81.5 | 68.0 | 58.0 | 100 | 81.5 | 85.0 |
| SD-C-NR >28.5 | 87.5 | 80.0 | 70.0 | 100 | 87.5 | 88.0 |
| MCV + MRV | 97.0 | 94.0 | 94.0 | 100 | 97.0 | 81.0 |
| MCV + SD-C-NR | 98.5 | 100 | 94.0 | 100 | 98.5 | 82.0 |
| MRV + SD-C-NR | 96.0 | 93.0 | 94.0 | 100 | 96.0 | 74.0 |
| MCV + MRV + SD-C-NR | 99.5 | 100 | 98.0 | 100 | 99.5 | 82.0 |
Discussion
The aim of screening for thalassemia and hemoglobinopathies in Southeast Asia is to offer carrier testing to the population before they have child [2, 20]. In the present study, we demonstrated the effectiveness combination parameters for thalassemia carrier screening in 200 various thalassemia syndromes. The identification of various types of thalassemia in this group of Thai participants (Table 1) confirms a high prevalence and a diverse heterogeneity of this genetic disorder in Southeast Asia and underlines the need for an appropriate and highly sensitive screening strategy. Hb E was the most common hemoglobinopathy among Southeast Asian populations [21] and subsequently β-thalassemia and α-thalassemias were detected at different frequencies.
A flow cytometry-based as an advanced technology in automated hematology analyzer has enabled us to study the heterogeneity of red cell parameters in α-and β-thalassemias [22–24]. The reduction of red blood cell indices such as MCV, MCH, and mean corpuscular hemoglobin concentration (MCHC) are the significant indicators for abnormal red cells in thalassemias [25–27]. MCV and MCH are used as the first screening tools for thalassemia trait since MCV and MCH are decreased in most thalassemias. It is noteworthy that the combined MCV and MCH method provided better sensitivity than that obtained using the MCV or MCH alone. For Hb E heterozygotes, however, many had normal RBC indices and, therefore, had negative results in MCV and MCH screening. The false negative result with MCV and MCH screening for Hb E carrier is unacceptable, especially when a population screened is known to have a high prevalence of Hb E, as is true for Southeast Asian populations. A reduction in MCV and MCH values also was noted in women with other mild forms of thalassemia, including α+-thalassemia, Hb CS, and Hb PS. Other investigators found similar results in a Chinese population [28, 29].
The results of the present study indicate the sensitivity of the MCV screening protocol was improved greatly (to 100.0 %) when it was used in combination with MRV and SD-C-NR. Based on the results of our study, we strongly recommend the use of the electronic red blood cell and reticulocyte counting for screening Southeast Asian populations to be able to identify and give proper advice to thalassemia carriers. It is interesting that the best screening result with 100.0 % sensitivity and 72.0 % specificity, were obtained with a combined MCV, MRV and SD-C-NR parameters (Table 4).
Conclusion
According to the present study, our screening method consisting of a combination of MCV, MRV and SD-C-NR parameters, is an attractive screening procedure. It is simple, reliable, cost-effective, and used only in one automated hematology analyzer, therefore requiring no extra labor. We propose this study protocol for the first screening to use for prevention and control of severe thalassemia in the provincial hospitals in Thailand. Using this method might provide earlier flagging of the thalassemia carriers, which through genetic counseling, could help reduce the incidence of thalassemia diseases in Southeast Asian communities.
Acknowledgments
We sincerely thank Naresuan University for financial support and also thank all co-workers for their blood collection and technical assistance at Medical Technology laboratory Unit, Sawanpracharak Hospital. We sincerely thank who the patients participated in this study and Asst. Prof. Dr. Ronald A. Markwardt, Burapha University, for his critical reading and correcting of the manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors have no conflict of interest to report.
References
- 1.Weatherall DJ. The global problem of genetic disease. Ann Hum Biol. 2005;32:117–122. doi: 10.1080/03014460500075480. [DOI] [PubMed] [Google Scholar]
- 2.Fucharoen S, Winichagoon P, Thonglairoam V, Siriboon W, Siritanaratkul N, Kanokpongsakdi S, et al. Prenatal diagnosis of thalassemia and hemoglobinopathies in Thailand: experience from 100 pregnancies. Southeast Asian J Trop Med Public Health. 1991;22:16–29. [PubMed] [Google Scholar]
- 3.Weatherall DJ. The thalassaemias. BMJ. 1997;314:1675–1678. doi: 10.1136/bmj.314.7095.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Wasi P. Data Center of Infectious Diseases and Carriers of Diseases. Thalassemia. http://webdb.dmsc.moph.go.th/ifc_nih/a_nih_1_001c.asp?info_id=403. (Thai version).
- 5.Thalassemia Foundation of Thailand. Clinical practice guidelines for diagnosis and management of thalassemia syndromes. 2014. http://www.thalassemia.or.th/thal-book/CPG_Thalassemia_2014-cover.pdf (Thai version).
- 6.The thalassemia working party of the BCSH general haematology task force. Guideline for investigation of the α- and β-thalassemia traits. J Clin Pathol. 1994;47:289–95. [DOI] [PMC free article] [PubMed]
- 7.A working party of the general haematology task force of the British Committee for standards in haematology. The laboratory diagnosis of haemoglobinopathies. Br J Haematol. 1998;101:783–92.
- 8.Silvestroni E, Bianco I. A highly cost effective method of mass screening for thalassemia. Br Med J (Clin Res Ed). 1983;286:1007–1009. doi: 10.1136/bmj.286.6370.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kattamis C, Efremov G, Pootrakul S. Effectiveness of one tube osmotic fragility test screening in detecting β-thalassemia trait. J Med Genet. 1981;18:266–270. doi: 10.1136/jmg.18.4.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Thool AA, Walde MS, Shrikhande AV, Talib VH. A simple screening test for detection of heterozygous β-thalassemia. Indian J Pathol Microbiol. 1998;41:423–426. [PubMed] [Google Scholar]
- 11.Frischer H, Bowman JE. Hemoglobin E an oxidatively unstable mutation. J Lab Clin Med. 1975;35:531–539. [PubMed] [Google Scholar]
- 12.Fucharoen G, Sanchaisuriya K, Sae-ung N, Dangwibul S, Fucharoen S. A simplified screening strategy for thalassemia and haemoglobin E in rural communities in south-east Asia. Bull World Health Organ. 2004;82:364–372. [PMC free article] [PubMed] [Google Scholar]
- 13.Lewis SM, Bain BJ, Bates I. Dacie and Lewis. Practical haematology. 9. London: Churchill Livingstone; 2001. [Google Scholar]
- 14.Bio-Rad. Standard Operating Procedure (SOP) for Sebia capillary electrophoresis; 2011. http://www.bio-rad.com/LifeScience/pdf/Bulletin_2651.pdf. Accessed 14 Apr 2014.
- 15.Fucharoen G, Fucharoen S. Rapid and simultaneous nonradioactive method for detecting α-thalassemia 1 (SEA type) and Hb Constant Spring genes. Eur J Haematol. 1994;53:186–187. doi: 10.1111/j.1600-0609.1994.tb00671.x. [DOI] [PubMed] [Google Scholar]
- 16.Sanchaisuriya K, Fucharoen G, Fucharoen S. Hb Paksé [(α2) codon 142 (TAA → TAT or Term → Tyr)] in Thai patients with EABart’s disease and Hb H disease. Hemoglobin. 2002;26:227–235. doi: 10.1081/HEM-120015026. [DOI] [PubMed] [Google Scholar]
- 17.Fucharoen S, Sanchaisuriya K, Fucharoen G, Panyasai S, Devenish R, Luy L. Interaction of hemoglobin E and several forms of α-thalassemia in Cambodian families. Haematologica. 2003;88:1092–1098. [PubMed] [Google Scholar]
- 18.Fucharoen S, Fucharoen G, Sriroongreung W, Laosombat V, Jetsrisuparb A, Prasatkaew S, et al. Molecular basis of β-thalassemia in Thailand: analysis of β-globin gene mutations using the polymerase chain reaction. Hum Genet. 1989;84:41–46. doi: 10.1007/BF00210668. [DOI] [PubMed] [Google Scholar]
- 19.Siriratmanawong N, Fucharoen G, Sanchaisuriya K, Ratanasiri T, Fucharoen S. Simultaneous PCR detection of β-thalassemia and α-thalassemia 1 (SEA type) in prenatal diagnosis of complex thalassemia syndrome. Clin Biochem. 2001;34:377–380. doi: 10.1016/S0009-9120(01)00250-8. [DOI] [PubMed] [Google Scholar]
- 20.Fucharoen S, Winichagoon P. Thalassemia in Southeast Asia: problem and strategy for prevention and control. Southeast Asian J Trop Med Public Health. 1992;23:647–655. [PubMed] [Google Scholar]
- 21.Fucharoen G, Fucharoen S, Sanchaisuriya K, Sae-Ung N, Suyasunanond U, Sriwilai P, et al. Frequency distribution and haplotypic heterogeneity of βE-globin gene among eight minority groups of northeast Thailand. Hum Hered. 2002;53:18–22. doi: 10.1159/000048600. [DOI] [PubMed] [Google Scholar]
- 22.Kim YR, Ornstein L. Isovolumetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry. Cytometry. 1983;3:419–427. doi: 10.1002/cyto.990030606. [DOI] [PubMed] [Google Scholar]
- 23.Tycko DH, Metz MH, Epstein EA, Grinbaum A. Flow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentration. Appl Opt. 1985;24:1355. doi: 10.1364/AO.24.001355. [DOI] [PubMed] [Google Scholar]
- 24.Mohandas N, Kim YR, Tycko DH, Orlik J, Wyatt J, Groner W. Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering. Blood. 1986;68:506–513. [PubMed] [Google Scholar]
- 25.Fucharoen S, Winichagoon P, Thonglairuam V, Siriboon W, Sae-Ngow B. Laboratory diagnosis for thalassemia. Ann Acad Med Singapore. 1989;18:424–430. [PubMed] [Google Scholar]
- 26.Bunyaratvej A, Fucharoen S, Greenbaum A, Mohandas N. Hydration of red cells in alpha and beta thalassemias differs. A useful approach to distinguish between these red cell phenotypes. Am J Clin Pathol. 1994;102:217–222. doi: 10.1093/ajcp/102.2.217. [DOI] [PubMed] [Google Scholar]
- 27.d’Onofrio G, Chirillo R, Zini G, Caenaro G, Tommasi M, Micciulli G. Simultaneous measurement of reticulocyte and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia. Blood. 1995;85:818–823. [PubMed] [Google Scholar]
- 28.Ma ES, Chan AY, Ha SY, Lau YL, Chan LC. Thalassemia screening based on red cell indices in the Chinese. Haematologica. 2001;86:1310–1311. [PubMed] [Google Scholar]
- 29.Chan LC, Ma SK, Chan AYY, Ha SY, Waye JS, Lau YL, et al. Should we screen for globin gene mutations in blood samples with mean corpuscular volume (MCV) greater than 80 fL in areas with a high prevalence of thalassemia? J Clin Pathol. 2001;54:317–320. doi: 10.1136/jcp.54.4.317. [DOI] [PMC free article] [PubMed] [Google Scholar]