Diagnostic Value of Non-Invasive Prenatal Screening of β-thalassemia by Cell Free Fetal DNA and Fetal NRBC

Author(s): Nadia Shafei, Mohammad Saeed Hakhamaneshi*, Massoud Houshmand, Siavash Gerayeshnejad, Fardin Fathi, Sardar Sharifzadeh.

Journal Name: Current Molecular Medicine

Volume 19 , Issue 2 , 2019

  Journal Home
Translate in Chinese
Submit Manuscript
Submit Proposal


Background: Beta thalassemia is a common disorder with autosomal recessive inheritance. The most prenatal diagnostic methods are the invasive techniques that have the risk of miscarriage. Now the non-invasive methods will be gradually alternative for these invasive techniques.

Objective: The aim of this study is to evaluate and compare the diagnostic value of two non-invasive diagnostic methods for fetal thalassemia using cell free fetal DNA (cff-DNA) and nucleated RBC (NRBC) in one sampling community.

Methods: 10 ml of blood was taken in two k3EDTA tube from 32 pregnant women (mean of gestational age = 11 weeks), who themselves and their husbands had minor thalassemia. One tube was used to enrich NRBC and other was used for cff-DNA extraction. NRBCs were isolated by MACS method and immunohistochemistry; the genome of stained cells was amplified by multiple displacement amplification (MDA) procedure. These products were used as template in b-globin segments PCR. cff-DNA was extracted by THP method and 300 bp areas were recovered from the agarose gel as fetus DNA. These DNA were used as template in touch down PCR to amplify b-globin gen. The amplified b-globin segments were sequenced and the results compared with CVS resul.

Results: The data showed that sensitivity and specificity of thalassemia diagnosis by NRBC were 100% and 92% respectively and sensitivity and specificity of thalassemia diagnosis by cff-DNA were 100% and 84% respectively.

Conclusion: These methods with high sensitivity can be used as screening test but due to their lower specificity than CVS, they cannot be used as diagnostic test.

Keywords: Prenatal diagnosis, β-thalassemia, screening, NRBC, Cff-DNA, non-invasive.

Fucharoen S, Winichagoon P. Hemoglobinopathies in southeast Asia. Hemoglobin 1987; 11(1): 65-88.
Smith CH. Smith’s blood diseases of infancy and childhood. Saint Louis: Mosby 1978.
Giardina P, Forget B. Thalassemia syndromes. Hematol Basic Principl Pract 2008; 5: 535-63.
Rachmilewitz EA, Giardina PJ. How I treat thalassemia. Blood 2011; 118(13): 3479-88.
Ghotbi N, Tsukatani T. Evaluation of the national health policy of thalassaemia screening in the Islamic Republic of Iran. East Mediterr Health J 2005; 11(3): 308-18.
Akolekar R, Beta J, Picciarelli G, Ogilvie C, d’Antonio F. Procedure‐related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta‐analysis. Ultrasound Obstet Gynecol 2015; 45(1): 16-26.
Berry SM, Stone J, Norton ME, Johnson D, Berghella V. Medicine SfM-F. Fetal blood sampling. AJOG 2013; 209(3): 170-80.
Benn P, Cuckle H, Pergament E. Non‐invasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet Gynecol 2013; 42(1): 15-33.
Bianchi DW, Flint AF, Pizzimenti MF, Knoll J, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci USA 1990; 87(9): 3279-83.
Lo YD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. The Lancet 1997; 350(9076): 485-7.
Wright CF, Burton H. The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Hum Reprod Update 2009; 15(1): 139-51.
Xue X, Teare MD, Holen I, Zhu YM, Woll PJ. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta 2009; 404(2): 100-4.
Jorgez CJ, Bischoff FZ. Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification. Fetal Diagn Ther 2009; 25(3): 314-9.
Finning K, Martin P, Summers J, Massey E, Poole G, Daniels G. Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study. BMJ 2008; 36(7648): 816-8.
Legler TJ, Liu Z, Mavrou A, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007; 27(9): 824-9.
Dhallan R, Au W-C, Mattagajasingh S, et al. Methods to increase the percentage of free fetal DNA recovered from the maternal circulation. JAMA 2004; 291(9): 1114-9.
Xu X-P, Gan H-Y, Li F-X, et al. A method to quantify cell-free fetal DNA fraction in maternal plasma using next generation sequencing: its application in non-invasive prenatal chromosomal aneuploidy detection. PLoS One 2016; 11(1): e0146997.
Kitagawa Y, Sugihara R. Genetic chromosome test management system, test management server, client terminal, genetic chromosome test management method, and program. Google Patents 2016.
Hudecova I, Chiu RW. Non-invasive prenatal diagnosis of thalassemias using maternal plasma cell free DNA. Best Pract Res Clin Obstet Gynaecol 2017; 39: 63-73.
Hahn S, Zhong XY, Holzgreve W. Recent progress in non-invasive prenatal diagnosis. Paper presented at. Semin Fetal Neonatal Med 2008; 13(2): 57-62.
Keshavarz Z, Moezzi L, Ranjbaran R, et al. Evaluation of a modified DNA extraction method for isolation of cell-free fetal DNA from maternal serum. AJMB 2015; 7(2): 85.
Douglas GW, Thomas L, Carr M, Cullen NM, Morris R. Trophoblast in the circulating blood during pregnancy. AJOG 1959; 78: 960-73.
Hahn S, Sant R, Holzgreve W. Fetal cells in maternal blood: current and future perspectives. Mol Hum Reprod 1998; 4(6): 515-21.
Beaudet AL. Using fetal cells for prenatal diagnosis: history and recent progress. Paper presented at: American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2016.
Cheng W-L, Hsiao C-H, Tseng H-W, Lee T-P. Noninvasive prenatal diagnosis. Taiwan J Obstet Gynecol 2015; 54(4): 343-9.
Hatt L, Brinch M, Singh R, et al. Characterization of fetal cells from the maternal circulation by microarray gene expression analysis-Could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis? Fetal Diagn Ther 2013; 35(3): 218-27.
D'Souza E, Ghosh K, Colah R. A comparison of the choice of monoclonal antibodies for recovery of fetal cells from maternal blood using FACS for noninvasive prenatal diagnosis of hemoglobinopathies . Cytometry Part B: Clinical Cytometry. 2009; 76(3): 175-80.
Samura O, Sekizawa A, Zhen DK, Falco VM, Bianchi DW. Comparison of fetal cell recovery from maternal blood using a high density gradient for the initial separation step: 1.090 versus 1.119 g/ml. Prenat Diagn 2000; 20(4): 281-6.
Prieto B, Alonso R, Paz A, et al. Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient. Prenat Diagn 2001; 21(3): 187-93.
avanagh D, Kersaudy-Kerhoas M, Dhariwal R, Desmulliez M. Current and emerging techniques of fetal cell separation from maternal blood. Chromatogr B 2010; 878(22): 1905-11.
Ponnusamy S, Mohammed N, Ho S, et al. In vivo model to determine fetal-cell enrichment efficiency of novel noninvasive prenatal diagnosis methods. Prenat Diagn 2008; 28(6): 494-502.
Di Naro E, Ghezzi F, Vitucci A, et al. Prenatal diagnosis of β-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 2000; 6(6): 571-4.
Brittain T. Molecular aspects of embryonic hemoglobin function. Mol Aspects Med 2002; 23(4): 293-42.
Han JY, Kim KH, Park JI, Kim IH, Je GH. Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture. J Korean Med Sci 2001; 16(2): 145-9.
Peng W, Takabayashi H, Ikawa K. Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis. Eur J Obstet Gynecol Reprod Biol 2007; 131(1): 13-20.
Normand E, Qdaisat S, Bi W, et al. Comparison of three whole genome amplification methods for detection of genomic aberrations in single cells. Prenat Diagn 2016; 36(9): 823-30.
Chiu RW, Lau TK, Leung TN, Chow KC, Chui DH, Lo YD. Prenatal exclusion of β thalassaemia major by examination of maternal plasma. Lancet 2002; 360(9338): 998-1000.
Li Y, Di Naro E, Vitucci A, Zimmermann B, Holzgreve W, Hahn S. Detection of paternally inherited fetal point mutations for β-thalassemia using size-fractionated cell-free DNA in maternal plasma. JAMA 2005; 293(7): 843-9.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [105 - 111]
Pages: 7
DOI: 10.2174/1566524019666190226124135
Price: $58

Article Metrics

PDF: 33
PRC: 1