A Novel System for Newborn Screening of Thalassemia and Hemoglobinopathies Using Capillary Electrophoresis Is Superior Than Isoelectric Focusing

Abstract

Introduction:Thalassemia (thal) and hemoglobinopathies (Hb variants) is highly prevalent in Thailand and nearly all Southeast Asian countries. In developed countries such as US and European countries, a newborn screening program for thal and hemoglobinopathies (NBS-Hb) has been incorporated in a routine healthcare to detect those with abnormal Hb in particular Hb S (Sickle cell disease) to provide early intervention for affected patients. Application of detecting patients and carriers could be useful for many developing countries such as Thailand as a part of our prevention and control program at a national level. At presence, isoelectric focusing (IEF) is a widely used platform for NBS-Hb, however a new capillary electrophoresis (CE) has recently been developed. It is of interest to evaluate whether which system provides a better efficiency under a circumstance with various types of thal and Hb variants.Objectives:To evaluate whether IEF or CE, is more effective for Hb screening at neonatal period and to develop new diagnostic criterion for different types of Hb variants in ThailandMethods:After informed consent by parent, the dried blood spot (DBS) samples were collected by heel prick puncture from 2-day old babies and processed in two Hb screening methods; IEF & ISOSCAN RESOLVE® SYSTEM Neonatal Hemoglobin Kits (Perkin Elmer, Turku, Finland) and Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). These DBS were also extracted gDNA used for gold standard molecular method covering >98% of common globin mutations found in Thailand; 7-deletional α-thal GAP-PCR, 6-non-deletional α-thal ARMS-PCR, 16 mutations of β-thal ARMS-PCR, PCR-RFLP by Mnl I restriction cleavage for Hb E disorder and/or DNA sequencing. The statistics analysis was performed by PASW 18 Software (SPSS Inc, 2015) to analyze the detection efficiency of IEF vs. CE. Cut-off levels of Hb variants were also determined by using receiver operating characteristic (ROC) curve to set up diagnostic criterion.Results: The study recruited 1,213 healthy newborns (48.4% male and 51.6% female) with average 38 weeks of gestational age (GA). The profiles of IEF pattern and CE histogram were analyzed. Hb E was the most common Hb variants of β-globin gene found and Hb Bart's (γ4) was indicative of α-thal, both were confirmed by molecular methods. Comparison between the efficiency of IEF vs. CE on detecting Hb variants showed a higher efficiency of CE on Hb Bart's detection for most α-thal carriers and on Hb E detection in Hb E trait than those of IEF for sensitivity, specificity, and accuracy as shown in Table 1. There was no difference between two platforms on Hb H disease, homozygous Hb E and β-thal/Hb E. In order to set up cut-off levels of Hb Bart's, Hb E and Hb A for each globin genotypes by ROC curve analysis, the cut-off levels provided from CE seemed to be more clear and discriminative than those of IEF as shown in Table 2. The cut-off levels of Hb Bart's of ≥7.40%, ≥0.75%, ≥0.45%, and ≥0.10% were suggested different α-globin abnormalities; Hb H, αº-, non-deletional and deletional α+-thal carrier, respectively. Detection of Hb E of ≥0.25%, ≥4.85%, and ≥0.95% were suggestive of Hb E carrier, Hb E homozygote, and β-thal/Hb E, respectively. Interestingly, we can screen for β-thal carriers using a lower Hb A level (≤10.35%) and Hb A/Hb F ratio (≤0.115). This data was analyzed from 11 β-thal carrier newborns confirmed by DNA testing compared to 148 GA-sex matched newborn controls with normal β-globin genotype. These cut-off levels based on CE were powerful on detecting thal diseases (Hb H and β-thal/Hb E) and carriers of Hb E, αº- and non-deletional α+-thal. However, our screening criterions still have a limitation on fully detecting deletional α+-thal and β-thal traits.Conclusions:Our data suggested that neonatal CE platform is more effective system for Hb screening with excellent efficiency and precision to detect Hb variants compared to conventional IEF. Using NBS-Hb by CE could successfully screen for all important thal carriers in one simple test. Implementing our NBS-Hb to the national level would be simplest way to generate a national registry that can be linked to our national health database of severe diseases and carriers. By applying this approach into a national scale might be the only measure to effectively control the health burden of thal and hemoglobinopathies in Thailand where provides high prevalence of defective globin genes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.