A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

OBJECTIVES: Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. METHODS: We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, ß-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. RESULTS: The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. CONCLUSIONS: The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α0-thalassemia--SEA,--THAI, and--CR type deletions.

Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α0-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α0-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α0-thal--CR,--THAI,--SEA, and the wild-type α-globin gene had specific peak heights at mean melting temperature (Tm) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α0-thal--SEA,--THAI,--CR obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart's hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.

Diagnosis of α0-thalassemia Chiang Rai (--CR) deletion by melt curve analysis in Northern Thailand.

A large novel 44.6 kb deletion named α0-thalassemia Chiang Rai (--CR) was first described in the individuals with uncommon Hb Bart's hydrops fetalis and HbH disease. This study aimed to develop a real-time gap PCR and melt curve analysis for the detection of --CR and investigate its frequency in northern Thailand. Among 4,952 blood samples, the assay was performed in 525 samples with a mean corpuscular volume (MCV) < 80 fL, HbA2 < 3.5%, HbA2+E < 25%, and negative for common deletional α0-thalassemia --SEA and --THAI. The developed method showed Tm values of 85.8 ± 0.0 °C and 91.5 ± 0.1 °C, which were specific for --CR and wild-type alleles, respectively. Nine (0.18% of 4,952 or 1.71% of 525) were positive for --CR, in which two were HbH disease and the rest were heterozygous for --CR. This study demonstrated the success of real-time gap PCR with melt curve analysis for --CR diagnosis. Additionally, the prevalence of --CR in the northern Thai population was comparable to --THAI. Thus, this study implies the importance of --CR in northern Thailand. Moreover, the developed real-time gap PCR with melt curve analysis is simple and highly accurate, and may be considered as an additional tool for routine α0-thalassemia --CR diagnosis in this region.

Multiplex Quantitative Real-Time Polymerase Chain Reaction and High-Resolution Melting Analysis for Identification of a Couple At-Risk of Having a Newborn with Severe Thalassemia.

Many polymerase chain reaction (PCR)-based techniques have been used for routine diagnosis of α- and ß-thalassemias. However, most require a multi step of post-PCR processes that are time-consuming and labor-intensive procedures. This study reported the successful use of multiplex quantitative real-time PCR (qPCR), with high-resolution melting (HRM) analysis for diagnosis of two common deletional α0-thalassemia (α0-thal) and 15 common ß-thalassemia (ß-thal) mutations, in order to identify a couple at-risk of having a newborn with severe thalassemia in the northern region of Thailand. With this approach, 22 (7.2%) of 306 couples were diagnosed as being at-risk for having a child with severe thalassemia, including three homozygous α0-thal, five homozygous ß-thal and 14 Hb E (HBB: c.79G>A)/ß0-thal disease. Our findings indicated that multiplex qPCR with HRM is applicable for routine molecular diagnosis in order to identify a couple at-risk of having a newborn with severe thalassemia, especially in an endemic region.

Proficiency Testing Program for Hb E (HBB: c.79G>A) Screening in Thailand Using Lyophilized Hb E Control Materials.

The dichlorophenol-indophenol (DCIP) test and microcolumn chromatography are simple methods commonly used for screening of Hb E (HBB: c.79G>A) in Thailand. However, there is no proficiency testing (PT) program for these screening tests. Thus, the aim of this study was to evaluate an efficiency of lyophilized hemoglobin (Hb) control materials used in the established PT program for Hb E screening at the Associated Medical Sciences-Clinical Service Center (AMS-CSC), Chiang Mai University, Chiang Mai, Thailand. Three cycles of PT were performed from June 2018 to July 2019. In each cycle, five different types of control materials were provided to the participants. Each participant analyzed the control materials in the same manner as in their routine practices for Hb E screening. The results showed that the number of participants increased from 95 in the first cycle to 126 and 134 in the second and third cycles, respectively. The numbers of participants who used the DCIP screening test and reported the result correctly increased from 79 (85.87%) to 106 (89.08%) and 112 (89.60%), respectively. Whereas those who used the microcolumn chromatography method and reported correct results were decreased from 100.0 to 85.71 and 66.67%, respectively. Thus, lyophilized Hb, control materials can be used effectively for the PT program of Hb E screening test. However, the further improvement, especially in skills of Hb E analysis by microcolumn chromatography, is required for some participating laboratories.

Validation of Microarray for the Simultaneous Detection of Common α- and ß-Thalassemia Gene Mutations.

BACKGROUND: Methods for detecting the complex genetic characteristics of α- and ß-thalassemias are required for preventing and controlling the outbreak of new cases. METHODS: We evaluated the accuracy and practical utility of microarray for simultaneous detection of α- and ß-thalassemias. A total of 102 DNA specimens, which represented 25 different genotypes, were tested in parallel using the microarray and reference methods used in the thalassemia laboratory of the Associated Medical Sciences-Clinical Services Center (AMS-CSC), Chiang Mai, Thailand. RESULTS: A total of 100 (98.0%) DNA specimens were completely concordant between the microarray and reference methods, whereas discrepancies between the different methods were observed in only 2 DNA specimens with homozygous hemoglobin E (HbE). CONCLUSIONS: The microarray appeared to be a fast, easy to perform, and accurate method for simultaneous detection of α- and ß-thalassemias in Thailand and Southeast Asian countries. However, this technique needs to be improved and validated in a larger number of specimens with homozygous HbE before further routine laboratory use.

A Formula to Identify Potential Cases of ß-Thalassemia/HbE Disease Among Patients With Absent HbA, HbE >75% and HbF Between 5 and 15.

OBJECTIVE: To establish a simple formula to be used for discrimination between ß-thalassemia/hemoglobin E (ß-thal/HbE) and homozygous hemoglobin (Hb)E in specimens with absent hemoglobin (Hb)A, HbE of greater than 75%, and HbF between 5% and 15%. METHODS: We analyzed laboratory results from February 2015 through February 2018. Molecular analysis for diagnosis of ß-thal mutation and HbE was performed in specimens that contained HbE of greater than 75% and HbF from 5% to 15%, as measured by high-performance liquid chromatography (HPLC). HbA2 and HbF levels were also measured by capillary electrophoresis. Then, the formula (6 × HbA2 + HbF)/MCV was developed. RESULTS: The score of 0.9 or higher was found in all 19 ß-thal/HbE specimens (100%) and only 8 of 65 homozygous HbE specimens (12.3%). Also, the formula yielded 90.5% efficiency in identifying ß-thal/HbE disease, and the efficiency was found to be higher compared with when the HbA2 value of greater than 6% was used by itself (85.4%). CONCLUSION: The formula (6 × HbA2 + HbF)/MCV, with a cutoff point at 0.9, could identify the potential cases of ß-thal/HbE disease among patients with absent HbA, HbE of greater than 75%, and HbF between 5% and 15%.

HbE Level and Red Cell Parameters in Heterozygous HbE With and Without α0-Thalassemia Trait.

We compared hemoglobin (Hb) E levels and red cell parameters between heterozygous HbE with and without α0-thalassemia trait and also determine their appropriated cut-off points for differentiating these two groups. High performance liquid chromatography analysis results and mean levels of red blood cell (RBC) parameters, including RBC count, total Hb, hematocrit, MCV, MCH and MCHC of heterozygous HbE with α0-thalassemia trait (n = 183) and without α0-thalassemia trait (n = 1437) were reviewed and compared. The α0-thalassemia status in these samples was detected by real-time PCR with SYBR Green1 and high resolution melting analysis. Mean levels of HbE, total Hb, MCV, MCH and MCHC of heterozygous HbE with α0-thalassemia trait were significantly lower than those of heterozygous HbE without α0-thalassemia trait (P < 0.001). In addition, HbE level at a cut-off value of < 24% was superior to MCV (< 80 fL) and MCH (< 27 pg) for differentiating the heterozygous HbE with and without α0-thalassemia trait with 100% sensitivity and 87.2% specificity. Despite certain limitations of this study like missing RDW and reticulocyte counts, and not testing for α+-thalassemia and Hb Constant Spring, we conclude that the HbE level at a cut-off point of < 24% is a useful marker for initial discrimination between heterozygous HbE with and without α0-thalassemia trait.

Hematological Analysis in Thai Samples With Deletional and Nondeletional HbH Diseases.

OBJECTIVES: To compare hematological parameters between deletional and nondeletional HbH diseases, and to investigate the correlation between HbH levels and hematological parameters within these 2 groups. METHODS: Samples of 43 deletional HbH diseases, which included 39 --SEA/-α3.7, 4 - -SEA/-α4.2, and 22 nondeletional HbH diseases (- -SEA/αcsα), were used in this study. Correlations between HbH levels and hematological parameters within these 2 groups were analyzed. RESULTS: The deletional HbH disease had higher levels of RBC counts, total Hb, pack cell volume (PCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), HbA, and HbA2 than did the nondeletional HbH disease. A negative correlation between HbH and RBC counts was detected in the group of deletional HbH disease, while a positive correlation between HbH and RBC counts, total Hb, and PCV was found in the group of nondeletional HbH disease. CONCLUSIONS: These results reflected that samples with nondeletional HbH showed more anemic features than those with the deletional HbH.

Proficiency testing program for hemoglobin E, A2 and F analysis in Thailand using lyophilized hemoglobin control materials.

BACKGROUND: There is no external quality assessment (EQA) program for hemoglobin analysis that uses lyophilized hemoglobin control materials with HbA2/E in levels as high as those found in people with the ß-thalassemia trait, HbE trait, ß-thalassemia/HbE disease or homozygous HbE; these are all found frequently in the southeast Asian population. The aim of this study was to evaluate the efficiency of the control materials used in the established proficiency testing (PT) program at the Associated Medical Sciences-Clinical Service Center (AMC-CSC), Chiang Mai University, Chiang Mai, Thailand. METHODS: The PT program for Hb analysis and the thalassemia interpretation was established in compliance with ISO/IEC17043:2010. Three cycles per year were performed in 2015 and 2016. In each cycle, three different types of control material were provided to the participants. Each participant analyzed the control materials in the same manner as in their routine practices. Hb analysis results and their thalassemia interpretation codes were entered into the report form and sent back to AMC-CSC. RESULTS: The number of participants increased from 63 in 2015 to 76 in 2016. In addition, the number of participants who took part in all three cycles increased from 95.2% (60/63) in 2015 to 100% (76/76) in 2016. All participants reported the correct Hb measurement and type; however, misinterpretations in thalassemia diagnosis were noted. CONCLUSIONS: The lyophilized hemoglobin control materials prepared at AMC-CSC were used successfully in our PT program. However, the study results indicate the need for further improvement in thalassemia interpretation skills for laboratory staff.

Development of hemoglobin typing control materials for laboratory investigation of thalassemia and hemoglobinopathies.

BACKGROUND: To date, the hemoglobin (Hb) typing control materials for laboratory investigation of thalassemia with low (1.8%-3.2%) and high (4%-6%) levels of HbA2 are available but there are no Hb typing quality control materials for analysis of thalassemia and hemoglobinopathies which are highly prevalent in South-East Asian countries. The main aim of the present study was to develop the lyophilized Hb typing control materials for laboratory investigation of thalassemia and hemoglobinopathies that are commonly found in South-East Asia. METHODS: Erythrocytes of blood samples containing Hb Bart's, HbH, HbE, HbF, Hb Constant Spring (CS), Hb Hope, and Hb Q-Thailand were washed and dialysed with 0.85% saline solution. The erythrocytes were then lysed in 5% sucrose solution. The lyophilized Hb typing control materials were prepared by using a freeze drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized Hb was performed after the storage at -20 °C for 1 year and also after reconstitution and storage at 4 or -20 °C for 30 days. In addition, the Hb analysis was compared between the three different methods of HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE). RESULTS: Following a year of storage at -20 °C, the HPLC chromatograms of lyophilized Hb typing control materials showed similar patterns to the equivalent fresh whole blood. The stability of reconstituted Hb typing control materials was also observed through 30 days after reconstitution and storage at -20 °C. Moreover, the Hb typing control materials could be analyzed by three methods, HPLC, LPLC and CE. Even a degraded peak of HbCS was found on CE electropherogram. CONCLUSIONS: The lyophilized Hb typing control materials could be developed and used as control materials for investigation of thalassemia and hemoglobinopathies.

Red Cell Indices and Formulas Used in Differentiation of ß-Thalassemia Trait from Iron Deficiency in Thai Adults.

ß-Thalassemia (ß-thal) and iron deficiency cause most microcytic anemias. Red cell indices and formulas have been established as simple, fast, and inexpensive in discrimination between these two hematological disorders in school children. However, whether these formulas could be applied to diagnose ß-thal trait and iron deficiency in adult Thai subjects is unclear. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and nine formulas (RDW/RBC, RDW Index, Sirdah, Green and King, Mentzer, England and Fraser, Ehsani, Srivastava and Shine and Lal). Their sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV), efficiency, and Youden's Index were analyzed in 102 ß-thal trait and 64 iron deficiency adult Thai subjects. The RDW/RBC formula proved to be the most reliable index as they had 100.0% specificity and PPV and the highest efficiency (94.58%) and Youden's Index (91.18%), as well as high sensitivity (91.18%) and NPV (87.67%). Therefore, this formula could be used in initial discrimination of ß-thal trait from iron deficiency in adult Thai subjects.

Detection of Hb Constant Spring (HBA2: c.427T>C) Heterozygotes in Combination with ß-Thalassemia or Hb E Trait by Capillary Electrophoresis.

Hb Constant Spring (Hb CS; HBA2: c.427T>C) is often missed by routine laboratory testing as its mRNA as well as gene product are unstable and presented at a low level in peripheral blood. This study aimed to analyze the efficacy of capillary electrophoresis (CE) for detecting and quantifying of Hb CS in ß-thalassemia (ß-thal) trait or Hb E (HBB: c.79G>A) trait samples with reduced ß-globin chain expression. Thalassemia diagnostic data were reviewed in 2524 blood samples that were submitted to the laboratory of the Associated Medical Sciences Clinical Service Center, Chiang Mai, Thailand for hemoglobinopathy and thalassemia diagnosis. DNA analysis for Hb CS was performed in 322 ß-thal trait and 397 Hb E trait samples using the amplification refractory mutation system (ARMS). The CE electropherogram of Hb CS at zone 2 was observed in all five samples with ß-thal trait and nine samples with Hb E trait with levels varying from 0.1-2.8 and 0.1-2.3%, respectively. Thus, the CE method proved useful for screening of Hb CS in samples with ß-thal trait or Hb E trait, which is essential for providing accurate diagnosis, genetic counseling, prevention and control programs of Hb H-CS disease.

Detection of compound heterozygous of hb constant spring and hb q-Thailand by capillary electrophoresis and high performance liquid chromatography.

A capillary electrophoresis (CE) has proven to be superior to a high performance liquid chromatography (HPLC) in the detection of hemoglobin Constant Spring (Hb CS). Thus the aim of this study was to analyze the efficacy of CE and HPLC for the detection of Hb CS in samples with compound heterozygous of Hb CS and Hb Q-Thailand. Hemoglobin analysis was performed in blood samples of 2 patients with compound heterozygous of Hb CS and Hb Q-Thailand by using HPLC and CE. The HPLC chromatogram and CE electrophoregram of the two techniques were compared. Hb CS was not found on HPLC chromatogram while Hb QA2 (α2 (QT)δ2), a derivative of Hb Q-Thailand, was presented at the retention time of 4.70-4.80 min and it was close to the retention time of Hb CS. On CE electrophoregram, Hb CS was presented at zone 2 (Z2) and it was distinctly separated from Hb QA2 which was presented at Z1. Therefore, CE was more efficient to the HPLC for diagnosis of compound heterozygous of Hb CS and Hb Q-Thailand.

Red cell indices and formulas used in differentiation of ß-thalassemia trait from iron deficiency in Thai school children.

Red cell indices and formulas have been established as simple, fast, and inexpensive means for discrimination between the ß-thalassemia (ß-thal) trait and iron deficiency. However, there were no reports of the diagnostic reliability of different red cell indices and formulas in discrimination of ß-thal trait from iron deficiency in the Thai population. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and eight formulas (Sirdah, Green & King, RDW Index, Menzler, England & Fraser, Ehsani, Srivastava, and Shine & Lal). Their sensitivity, specificity, positive and negative prognostic value and efficiency, were analyzed in 77 Thai school children, 21 with the ß-thal trait and 56 with iron deficiency. The Sirdah and Srivastava formulas proved to be the most reliable indexes as they had 100.0% sensitivity and negative predictive value, the highest efficiency (97.4%), and the highest Youden's Index value (96.4%). Therefore, these formulas could be used in initial discrimination of the ß-thal trait from iron deficiency in Thai school children.

Criteria for detection of alpha-thalassemia-1 Thai type deletion in routine laboratory.

BACKGROUND: Using a single tube multiplex real-time PCR with SYBR Green1 and high resolution melting (HRM) analysis in routine laboratory for detection of alpha-thalassemia-1 Southeast Asian (SEA) type deletion simultaneously with Thai type deletion is costly. METHODS: Criteria for laboratory testing of alpha-thalassemia-1 Thai type deletion were set as follow: (1) MCV < or = 75 fL and negative for alpha-thalassemia-1 SEA type deletion, beta-thalassemia or homozygous HbE; (2) HbE trait with HbE level < 21.5% and negative for a-thalassemia-1 SEA type deletion; or (3) Hb Bart's and HbH found on HPLC chromatogram but negative for alpha-thalassemia-1 SEA type deletion. RESULTS: From May 2010 to October 2012, the number of samples to detect for alpha-thalassemia-1 Thai type deletion decreased from 15,081 to 672 samples and the alpha-thalassemia-1 Thai type deletion trait was found in 7 samples (1.04%). CONCLUSIONS: The new testing algorithm can reduce number of samples for detection of alpha-thalassemia-1 Thai type deletion.

Hb A2/E levels found in co-inheritance with the α-thalassemia-1 - -(SEA)/type deletion and either Hb E or ß-thalassemia.

The α-thalassemia-1 (α-thal-1) Southeast Asian (- -(SEA)) type deletion, ß-thalassemia (ß-thal) and Hb E [ß26(B8)Glu→Lys, GAG>AAG] are the most common genetic disorders in Southeast Asian populations. Mean corpuscular volume (MCV) <80.0 fL with normal hemoglobin (Hb) is used for screening α- and ß-thal, and a Hb E level of less than 25.0% is used for predicting α-thal-1 in Hb E trait. Thus, levels of Hb, MCV and Hb A(2)/E were reviewed and compared between the SEA type deletion co-inherited with ß-thal trait (n = 61), with Hb E trait (n = 102) or homozygous Hb E (n = 13) and ß-thal trait (n = 636), Hb E trait (n = 544) or homozygous Hb E (n = 83), respectively. When comparing the values of all three analyzed hematological parameters, only the - -(SEA)/ß(E) values were shown to be significantly lower than those of Hb E trait. Furthermore, at a cut-off value of Hb A(2)/E of 21.54%, 95.0% of the - -(SEA)/ß(E) had Hb A(2)/E levels lower than this cut-off value, while 94.0% of Hb E trait had Hb A(2)/E at higher levels. Accordingly, the Hb A(2)/E level at 21.54% is the best indicator for predicting co-inheritance of the α-thal-1 - -(SEA)/ deletion and Hb E trait.