Development Of A Capillary Zone Electrophoresis Method For Rapid Determining Human Globin Chains

Background and PurposeHuman hemoglobin, a significant functional protein with the relative molecular weight of approximately 64.5 KDa, mainly exists in the erythrocytes as a transporter of oxygen and carbon dioxide in individuals. It is globular in structure, which form a shell around a central cavity including four oxygen-carrying heme groups each covalently bound a globin subunit. Human hemoglobin is a polypeptide tetrametric molecule encoded by two α-like chains (ζ or α) and two β-like chains (ε, γ,δ or β) non-covalently linked to each other. Due to the body’s various demands for oxygen in different development stages, the compositions of hemoglobin are characterized by expressing developmentally. During embryonic development, the ζ, α, ε or γ chains primarily contribute to embryonic Hbs, such as, Hb Gower 1 (ζ2ε2), Hb Gower 2 (α2ε2) and Hb Portland (ζ2γ2), which are the major hemoglobin productions in embryo. Furthermore, during fetal development, as the reduced ζ-, ε-globin synthesis and increased γ-globin gene expression, the major Hb type is HbF (α2γ2) with the minor HbA2 (α2δ2) fraction. Moreover, because of the second switch of hemoglobin production, y-globin gene closes at birth while P-globin gene expresses maximally after one year of birth and maintains lifelong. It is at this stage that the major Hb patterns are HbA (α2β2) and HbA(α2δ2).The hemoglobinopathies can be genetically classified as two distinct disease groups:thalassemias and abnormal hemoglobins. Thalassemias are hereditary recessive anemia characterized by insufficient production of α or β globin chains, which lead to the imbalance of human globin chains. According to the degree of α-and β-chain imbalance, thalassemias can be divided into the following three categories:(Ⅰ) Thalassemia carriers/trait. Subjects who carry mutations or deletions affecting the α or β globin gene on one chromosome, associated with minimal anemia, are said to have thalassemia trait. (Ⅱ) Thalassemia intermedia. Patients with thalassemia intermedia present later in life with variable degrees of anemia, which are mostly compound heterozygotes and some homozygotes. (Ⅲ) Thalassemia major. Individuals with this type of β-thalassemia are homozygotes or compound heterozygotes for β0 or β+ genes, who usually present within the first two years of life with severe anemia, requiring regular blood transfusions. Hb Bart’s hydrops foetails is a lethal form in which no alpha-globin is synthesized. Abnormal hemoglobins are a group of inherited hemolytic disorders associated with altered structures and functions, resulting from changes in the primary structure of the globin chains.In fact, phenotype studies play an important role in the diagnosis and prognosis of hemoglobin disorders despite the development of a number of molecular biology techniques. Several direct and indirect markers such as low mean corpuscular volume (MCV), low mean corpuscular hemoglobin (MCH), abnormal HbA2, increased HbF and presence of abnormal hemoglobins are used as complementary in diagnosis of thalassemia and hemoglobin variants. However, the complex relationship between phenotype and genotype lead to increase the difficulty in study of thalassemia. For a better understanding of thalassemia, the analysis and identification of globin chains is particularly important in the study of structural modifications, revealing the degree of a/non-a chain imbalance, identifying unknown Hb variants and monitoring globin gene therapies in animal experiment and related hematologic studies. To date, a variety of analytical methods have been reported for separation of globin chains, including immunoassay, gel electrophoresis, HPLC and RP-HPLC. However, the main drawbacks of these methods are labour intensive, time-consuming sample preparation and limited resolution. Recently, capillary electrophoresis (CE) is becoming an increasingly attractive separation technique for human globin chains due to its ability to separate charged compounds with high efficiency, low sample consumption, fast analysis times, and low solvent consumption. Some researchers have focused on the use of CZE with coated capillary for the separation and quantification of globin chains. Nevertheless, expensive cost of commercial permanently coated capillaries limits its application in clinical laboratories. CZE with dynamic modifiers in fused-silica capillary at acid pH is an alternative first line technique. In practice, there are still many problems to deal with:How to simplify the complex process and how to obtain a higher resolution in a relatively short period of time. Improvement of the performance of the capillary separation and optimization of the separation conditions in CZE may the two crucial points. Furthermore, a challenge to analysis of human globin chains is the selection of initial separation conditions and subsequent optimization of the appropriate experimental parameters for a capillary without a ready electrophoresis program.Here, we have developed a rapid, automated electrophoresis method for separation of α,β, and y globin chains from both newborn and adult samples without the need for complex sample pre-preparation. This CZE method plays a substantial role in analysis of thalassemia and hemoglobinopathies. Our experience in developing this method for the rapid separation of human globin chains could be of use for similar work.Materials and MethodsClinical samplesIn this study,24 samples from normal adult (4 cases), α-thalassemia silent (4 cases), α-thalassemia trait (4 cases), Hb H disease (4 cases), β-thalassemia carrier (4 cases) and β-TI/TM (4 cases) samples were used to develop the method. Additionally, one for each of four samples (normal adult, normal newborn, Hb H disease,β-TI/TM) were used to assess assay reproducibility. These samples were pre-typed by molecular genetic assay. For assay evaluation, a total of 310 clinical samples consisting of normal (n=58), α-thalassemia silent/trait (n=107, including 6 Hb WS and 1 Hb G-Honolulu), Hb H disease (n=39, including 1 Hb G-Honolulu), β-thalassemia carriers (n=43,including 2 HbE and 1 co-inheritance of α-thalassemia samples with Hb WS) and β-TI/TM (n=63) were obtained from EDTA-anticoagulated whole blood. Hemoglobin analysis was carried out using the high-performance liquid chromatography. Point mutations and deletions in the α-, β-,and γ-globin genes were detected using RDB assay while the deletional forms of a thalassemia were determined by gap-PCR.CZECZE was performed using a P/ACE MDQ Capillary Electrophoresis System equipped with a 70 cm x 50 cm uncoated fused-silica capillary at 25℃. The separation voltage was 19 KV and the UV detector was set at 214 mn. The electrophoresis buffer (100 mM sodium phosphate) was adjusted to pH 2.14 with TFA, and contained 0.25% PEG-6000 (Purity≥99.5%). Samples were pressure-injected for 3 s. Prior to separation,25 μl of whole blood was washed one time with saline to remove plasma (3000 r/min for 3min), and red cells were hemolyzed in 1 ml deionized water. The hemolysate was centrifuged for 10 min at 4000 r/min to remove cells debris.Statistical analysisMean values of the a/(3 area ratios between controls and patient groups were compared by ANOVA and ROC curves were plotted to evaluate the sensitivity and specificity and to determinate the appropriate cut-off values for predicting thalassemia patients. The area under the curve (AUC) was calculated to further investigate the ability of this method to discriminate thalassemia patients. Bivariate correlation analysis was conduct to determine the association between the area ratio of γ/αand HbF levels in the samples from P-TI/TM by using the spearman correlation coefficient. P-values<0.05 were considered significant, and analysis were carried out with SPSS 13.0 statistical analysis software (IBM, Armonk, NY, USA).Results and DiscussionMethod development and optimizationFor the initial separation of α,β and γ chain,8 samples from normal adult (n=4), Hb H disease (n=4) were analyzed. Compared with the normal samples, the peak eluting at 14.69 min was confirmed as α-globin as it was significantly reduced during separation of Hb H samples. Since γ chains contribute to HbF,52 Hb samples from β-TI/TM patients with different HbF levels (10%-95%) were used to verify the α, β and γ chain content. In the present work, the area of the peak at 12.97 min increased with higher HbF, in contrast with the peak area at 13.30 min which was reduced. Furthermore, an obvious positive correlation between the ratio of peak (eluted at 12.97 min)/a (a as a reference) and HbF levels was observed (R2=0.8318, P<0.01). Therefore, the peaks at 12.97 min and 13.30 min corresponded to γ and β chains, respectively.In order to shorten the time needed for analysis, red cell hemolysates were injected directly into capillaries without prior sample preparation. Although pH did not affect the migration time, a pH<3 resulted in sharper peaks and higher resolution. After preliminary experiments, a pH of 2.14 was chosen, since separation performed at this pH provided the best resolution of α- and β-globin chains. It has been reported that extreme pH and high phosphate buffer concentration (-50 mM) could dissociate the chains directly into α- and β-globin chain. In the present study,100 mM phosphate was used as a compromise between speed of analysis and resolution. Voltage and temperature were also important factors for the separation. As voltage increased, migration time was reduced, but so was the ability to resolve the two chains. Although a higher temperature enabled a faster migration time, abnormal peaks appeared and the slope of the baseline increased, which prevented efficient separation of the chains. The optimum voltage and temperature were 19 KV and 25℃, respectively.Hb samples consisting of washed red cells and whole blood from the same normal adult were injected into capillaries and resolved using the conditions described above, and all globin chains (pre-α,α and β) were effectively separated. The elution sequence was as follows:pre-P chain appeared at 12.38 min, followed by β-chain at 13.37 min, and a-chain at 14.47 min. Separation of samples without cell washingand plasma controlsshowed that the plasma was faster than P chain, co-elution with pre-β chain. The migration times of α-and β-chain in samples after simple pre-treatment were similar to those of fully prepared Hb samples. The slope of the baseline increased slightly due to the plasma peak, which could affect the quantitative analysis of pre-β-chain, but not the other two major peaks. With the newborn Hb sample, the pre-β peak was detected at 12.27 min, followed by the y peak at 12.97 min, and the β peak at 13.30 min, and the a-peak at 14.69 min. Hb G-Honolulu and Hb WS were also successfully separated and identified using this CZE method:αWS chain were eluted at 14.54 min, faster than normal a-chain, while aG Honolulu ckain were detected at 15.13 min, later than normal a-chain. These results are consistent with the HPLC analysis.Application studyIn order to evaluate the reproducibility of the migration time and peak areas in the detection of α,β and γ chains in different clinical samples, four known samples (normal adult, normal newborn, Hb H disease, P-TI/TM) were selected. Five sequential injections of these samples gave almost identical migration time and peak area data. The migration times of α-, β-and γ-chain from healthy samples were slightly earlier than those of β-TI/TM samples, but according to the instrument settings, the difference was<5% and is therefore acceptable. The coefficient of variation (CV) for migration times and areas ranged from 0.37% to 1.69% and 0.46% to 6.71%, respectively.P-values for the α/β area ratios between healthy control and diseased (a-thalassemia silent/trait, Hb H, β-thalassemia carrier and β-TI/TM) patient groups were<0.05. The mean α/β ratio of the normal control group was very close to a-thalassemia silent/trait and β-thalassemia patients.Based on a comparison of multiple results, ROC curves were plotted using data sets from 58 healthy controls,107 α-thalassemia silent/trait patients,39 Hb H disease patients,43 β-thalassemia carriers and 63 P-TI/TM patients. The predictive accuracy of the α/β ratio was assessed by calculating the AUC, which was 0.881 for α-thalassemia silent,0.887 for a-thalassemia trait,0.975 for Hb H disease,0.790 for P-thalassemia carrier and 1.000 for P-TI/TM samples. ROC analysis demonstrated that the optimal cut-off value for identifying Hb H disease from the α/β ratio was 0.949 (sensitivity= 97.4, specificity=100), and was 1.14 for β-TI/TM (sensitivity= 100, specificity=100; Fig 4b and c). For prediction of a-thalassemia silent, α-thalassemia trait (SEA) and β-thalassemia carrier, the sensitivity and specificity were 60%-90%.DiscussionIn this study, we developed a relatively rapid, automated and inexpensive CZE approach for separation of human globin chains without the need for prior sample preparation that may be suitable for separation and quantification of globin chains. Over the past 30 years various methods with varying degrees of speed and accuracy have been used for hemoglobin analysis in clinical laboratories, the most commonly used of which is HPLC, which can resolve most common hemoglobin variants and can accurately quantify HbA2 and F. However, Hb WS and Hb QS variants, which occur frequently in the Chinese population, are difficult to detect using this HPLC method. Other strategies based on CE modes coupled with various detection methods have emerged for determination of globin chains, including capillary isoelectric focusing electrophoresis (CIEF) and micellar electrokinetic capillary chromatography (MECC). These two methods are mainly used for the routine analysis of abnormal hemoglobins, which limits their role in the aid in diagnosis of thalassemia.Compared with other separation methods, the advantages of the CZE method developed in the present study are as follows:(Ⅰ) cost-effectiveness, good resolution and reproducibility. It was reported that the performance of coated capillaries deteriorates after several weeks of continuous electrophoresis and separation of 100 samples, although coated capillaries avoid protein sticking to the wall of the capillary, resulting in improved resolution. Furthermore, preparing a permanently coated capillary directly in the laboratory is complex and time-consuming. In the present study, PEG-6000 was used to minimize protein absorption, which gave high resolution and reproducibility at the cost of approximately $0.1 per sample. Meanwhile the CV of this methodology was very low, demonstrating assay good reproducibility. (Ⅱ) The expensive equipment and strict experimental conditions are not required, and the operation procedure relies on simple technical requirements, so it is particularly suitable for use in developing countries. (Ⅲ) Speed and convenience. The globin chains were denatured and separated from the heme groups in 17 min without sample pre-treatment. However,13 minutes for sample preparation before separation will obtain better resolution. The process of sample preparation was also simple compared with the long analytical time taken in globin precipitation and extraction in previous approaches. And the elution pattern of the sample without cell washing was similar to the simple prepared Hb solution. (IV) Most importantly, the analytical process can be completely automated with the CZE method.Since the globin chain synthesis abnormalities lead to unbalanced α,β globin chain, the area ratio of α/β was an effective marker for α-and β-thalassemia. To evaluate this marker, a total of 310 clinical samples were examined by CZE. Based on the α/β results of these samples, it shows significant different between the normal and patients by ANOVA (P<0.05). However, the mean ratios α/β of α-thalassemia silent/trait and β-thalassemia carrier are closer to that of normal compared to the difference between Hb H disease, β-thalassemia intermedia/major and normal, possibly because of the compensatory over-expression of the complementary strand or the degradation of excess globin chains. According to the ROC analysis that carried out to establish the most suitable cut-off value for thalassemia prediction, it had a high sensitivity and specificity in the procedure presented here for Hb H disease and β-thalassemia intermedia/major in our study. Additionally, the sensitivity and specificity of the proposed cut-off value for indicating a-thalassemia silent/trait and β-thalassemia carrier were lower, approximately 80%. As a result, specificity of this method for α-and β-thalassemia carrier was lower than globin as described previously. Therefore, this method might be considered as complementary to the other diagnosis tool for a-thalassemia silent/trait and P-thalassemia carrier. However, none of the methods can distinguish all the types of thalassemia up to now. Despite these limitations, for monitoring gene therapy or predicting for Hb H disease and β-thalassemia intermedia/major, the sensitivity and specificity of the present method would be sufficient.For abnormal hemoglobins, Hb WS and Hb G-Honolulu were detected in the CZE method, although HbE, Hb CS and Hb QS could not be discriminated, possibly due to degradation of the abnormal globin chains and/or the limited resolution of the CZE method. The next important step is to collect Hb variants to evaluate the sensitivity and accuracy of separation of abnormal hemoglobins.In the present study, we tried to dissociate globin chains from their heme groups before separation using urea and β-mercaptoethanol, however these reagents denatured and precipitated the proteins, which lowered the detection sensitivity and generated nonspecific peaks that masked the target peaks. Injecting an aqueous solution of hemoglobin into the capillary was found to give adequate resolution. We also tried to separate the two types (Gγ and Ay) of γ chains directly in running buffer, which contained 1% surfactant. However, there was no distinct peak detected over 40 min. Despite this caveat, the elution of α-, β-and y-chain is sufficient for routine clinical diagnosis.It has been reported a few strategies of analysis of thalassemia and hemoglobin variants. Analytical methods based on experimental conditions of each laboratory are diverse. Establishing a new method with optimal conditions of globin chains separation is time consuming. Here, we hope to have reference value for the similar work by summarizing the change of the separation behavior that affected by the main setting parameters and the composition of the running buffer.The excellent separation of α-,β-and γ-chain and identification of hemoglobin variants could make the CZE method suitable for the clinical diagnosis of thalassemia and abnormal hemoglobin variants. ROC analysis of the α/β area ratio showed a high sensitivity and specificity for indicating β-TI/TM and Hb H disease by using this method. Furthermore, CZE may prove useful for monitoring the effects P-thalassemia treatments, or for the separation of globin chains in gene therapy trials. In addition, CZE may elucidate structural modifications of globin chains for subsequent structural studies.