Background: Besides detecting hemoglobin (Hb) variants such as sickle cell disease for early preventive measure, newborn screening (NBS) program using Hb profile could identify other forms of thalassemia (thal) and Hb disorders. Due to continuous expression of fetal Hb (γ globin chains), the presence of Hb Bart's (γ4) at birth caused by unbalanced α to β like-globin synthesis have been used to screen for α-thal. So far, this NBS approach appears to be the only laboratory evaluation of α-thal screening for both carriers and Hb H disease since soon after birth Hb Bart's level would decline and we can only use DNA analysis to identify α-thal traits. Neonatal screening of αº-thal due to two missing α-globin genes (--/αα or -α/-α) from normal four copies and Hb H disease (3 α-globin genes lost; --/-α) could be achieved by using the percentage of Hb Bart's cutoffs from isoelectric focusing (Jindatanmanusan P et al. 2014) or capillary electrophoresis (Suksangpleng T et al. ASH 2017). However this approach has a major limitation to screen for α+-thal trait both deletional (-α/αα) and non-deletional (αTα/αα) types due to rather low sensitivity and specificity.
Objective: To develop a novel scheme for early detection of all types of α-thal using a combination of neonatal hematological parameters and Hb profiles at birth
Methods: After informed consent approved by the local ethical committee, 0.3-ml EDTA whole blood samples and dried blood spot were collected within 48 hrs after birth in newborn delivered at Siriraj Hospital by heel prick puncture. We measured the complete blood count (CBC) by using Unicel® DxHTM800 Coulter® Cellular Analysis System (Beckman Coulter, Brea, USA). The gDNA was also extracted for PCR-based molecular analysis to detect > 98% of α- and β-globin mutations mostly found in Thailand. Neonatal Hb profile was measured in qualitative and quantitative analysis using Capillarys 2 NEONAT FAST® (SEBIA, Évry, France). All data from each genotype groups were statistically analyzed using PASW statistics 18 Software (SPSS Inc, 2015).
Results: Total 732 healthy Thai neonates consisted of normal α-globin alleles (αα/αα; n=505, 69%), α+-thal carrier (-α/αα; n=139, 19%), non-deletional α-thal carrier (αTα/αα, n=33, 4.5%), α°-thal carrier (--/αα) and homozygous α+-thal (-α/-α; n=43, 5.9%), compound heterozygous α+-thal with non-deletional type (-α/αTα; n=8, 1%), and Hb H disease (--/-α; n=4, 0.6%). This population had normal β-globin genotype (ββ; n=533, 72.8%), Hb E carrier (βEβ; n=183, 25%) and homozygous Hb E (βEβE; n=16, 2.2%). Interestingly, red cell indices (MCV and MCH) and Hb Bart's levels were significantly altered due to different α-globin genotypes but not by Hb E coinheritance, gender, gestational age or history of neonatal jaundice. These suggested that the variation of red cell indices at birth is largely dependent on the number of expressed α-globin genes. Therefore we hypothesized that by using a combination of red cell indices and Hb Bart's level, we could improve the efficiency of α-thal screening at birth. Indeed, we have identified several cutoffs of Hb Bart's, MCV and MCH by using a ROC analysis that could differentiate individuals with different defective α-globin genes (data not shown). We have developed a range of cutoffs and in combination using either MCV or MCH as a first screening tool followed by Hb Bart's levels, the sensitivity and specificity of α-thal screening could significantly increase (Figure 1A and B). Moreover, we previously showed the 30% sensitivity and 94% specificity to detect α+-thal trait by using Hb Bart's cutoff alone. Interestingly, our approach can increase both sensitivity and specificity up to >90%. This was also true for other types of α-genotypes (Table 1). This novel approach has a significant impact on screening for both deletional and non-deletional α+-thal traits (-α and αTα).
Conclusion: For the first time, this novel combined screening using either MCV or MCH together with Hb Bart's could be used to detect all types of α-thal with a very high sensitivity (>85%) and specificity (100%). This approach would be useful to identify individuals with α-thal as a part of our national program for prevention and control of thalassemia disease currently operating in Thailand. Moreover our recommended screening flow described herein would be useful for other Southeast Asian countries where more than 30% of population is affected by α-thal.
Viprakasit:Agios: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.