Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR
-
Wenqian Song
Abstract
Background: Fetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR.
Methods: Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylated RASSF1A sequences of fetal origin in maternal plasma. Fetal ABO genotypes were determined by SYBR-based real-time PCR with allele-specific primers. The performance of the fetal ABO genotyping was assessed by the blood group serology results of the newborns.
Results: The fetal RASSF1A sequences were detectable in all the 73 plasma samples, which confirmed the successful extraction of cffDNA. The diagnostic accuracy of fetal ABO genotyping was 93.2%, in which the accuracy of fetal genotype OO, OA and OB was 100%, 83.3% and 96.8%, respectively.
Conclusions: We have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.
Acknowledgments
We thank all the individuals participated in this study, and all of our colleagues who helped collecting the blood samples. We thank Zhang Nianzhu and Ma Kexin for their technical assistance. We also thank Dr. Liang Wei who helped us on ABO gene sequencing.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: Dalian Municipal Commission of Health and Family Planning and Dalian Blood Center.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References
1. Eder A. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22:188–95.10.21307/immunohematology-2019-379Search in Google Scholar
2. Stiller RJ, Herzlinger R, Siegel S, Whetham JC. Fetal ascites associated with ABO incompatibility: case report and review of the literature. Am J Obstet Gynecol 1996;175:1371–2.10.1016/S0002-9378(96)70057-1Search in Google Scholar
3. Lo Y, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7.10.1016/S0140-6736(97)02174-0Search in Google Scholar
4. Bianchi DW. Fetal DNA in maternal plasma: the plot thickens and the placental barrier thins. Am J Hum Genet 1998;62:763–4.10.1086/301809Search in Google Scholar PubMed PubMed Central
5. Lo Y, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.10.1086/301800Search in Google Scholar PubMed PubMed Central
6. Chan KA, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92.10.1373/clinchem.2003.024893Search in Google Scholar PubMed
7. Lo YD, Chiu RW. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis. Clin Chem 2008;54:461–6.10.1373/clinchem.2007.100016Search in Google Scholar PubMed
8. Pertl B, Sekizawa A, Samura O, Orescovic I, Rahaim PT, Bianchi DW. Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats. Hum Genet 2000;106:45–9.10.1007/s004399900166Search in Google Scholar
9. Chim SS, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, et al. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci USA 2005;102:14753–8.10.1073/pnas.0503335102Search in Google Scholar PubMed PubMed Central
10. Chan KA, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211–8.10.1373/clinchem.2006.074997Search in Google Scholar PubMed
11. Muro T, Fujihara J, Imamura S, Nakamura H, Kimura-Kataoka K, Toga T, et al. Determination of ABO genotypes by real-time PCR using allele-specific primers. Leg Med 2012;14:47–50.10.1016/j.legalmed.2011.10.002Search in Google Scholar
12. Lo YD. Fetal DNA in maternal plasma: biology and diagnostic applications. Clin Chem 2000;46:1903–6.10.1093/clinchem/46.12.1903Search in Google Scholar
13. Lo YD, Chan KA, Sun H, Chen EZ, Jiang P, Lun FM, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2010;2:61ra91.10.1126/scitranslmed.3001720Search in Google Scholar
14. Meng J-L, Wang X-T, Wang Y, Yue Y-F, Wang X, Chen Z-J. Use of maternal plasma for non-invasive prenatal diagnosis of fetal ABO genotypes. Clin Chem Lab Med 2007;45:981–6.Search in Google Scholar
15. Zhou G-H, Shirakura H, Kamahori M, Okano K, Nagai K, Kambara H. A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products. Hum Mutat 2004;24:155–63.10.1002/humu.20052Search in Google Scholar
16. Okano DK, Uematsu C, Matsunaga H, Kambara H. Characteristics of selective polymerase chain reaction (PCR) using two-base anchored primers and improvement of its specificity. Electrophoresis 1998;19:3071–8.10.1002/elps.1150191805Search in Google Scholar
17. Yaku H, Yukimasa T, Nakano S, Sugimoto N, Oka H. Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem. Electrophoresis 2008;29:4130–40.10.1002/elps.200800097Search in Google Scholar
18. Okimoto R, Dodgson J. Improved PCR amplification of multiple specific alleles (PAMSA) using internally mismatched primers. Biotechniques 1996;21:20–2, 24, 26.10.2144/96211bm03Search in Google Scholar
19. Ficko T, Galvani V, Rupreht R, Dovc T, Rožman P. Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR – SSP method. Transfus Med 2004;14:425–32.10.1111/j.1365-3148.2004.00538.xSearch in Google Scholar
20. Nakamura S, Matsushita H, Nagai T, Sugie H, Furukawa M, Kurihara K, editors. DNA analysis of ABO blood group system detected by single-base nucleotide substitutions in a paternity case. International Congress Series. Amsterdam: Elsevier, 2003.10.1016/S0531-5131(02)00503-4Search in Google Scholar
21. Blumenfeld OO. Compilation of ABO alleles in BGMUT. March 2011.Search in Google Scholar
22. Yip SP. Single-tube multiplex PCR-SSCP analysis distinguishes 7 common ABO alleles and readily identifies new alleles. Blood 2000;95:1487–92.10.1182/blood.V95.4.1487.004k53_1487_1492Search in Google Scholar
23. Watanabe G, Umetsu K, Yuasa I, Suzuki T. Amplified product length polymorphism (APLP): a novel strategy for genotyping the ABO blood group. Hum Genet 1996;99:34–7.10.1007/s004390050306Search in Google Scholar PubMed
24. Stroncek D, Konz R, Clay M, Houchins J, McCullough J. Determination of ABO glycosyltransferase genotypes by use of polymerase chain reaction and restriction enzymes. Transfusion 1995;35:231–40.10.1046/j.1537-2995.1995.35395184280.xSearch in Google Scholar PubMed
25. Gassner C, Schmarda A, Nussbaumer W, Schonitzer D. ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers. Blood 1996;88:1852–6.10.1182/blood.V88.5.1852.1852Search in Google Scholar
26. Maeda K, Nakamura S, Murakami C, Irie W, Watanabe T, Sasaki C, et al. ABO genotyping by TaqMan assay and allele frequencies in a Japanese population. Leg Med 2013;15:57–60.10.1016/j.legalmed.2012.08.009Search in Google Scholar PubMed
27. de Silva M, Whittle M. Guidelines for blood grouping and red cell antibody testing during pregnancy. Transfus Med 1996;6:71–4.10.1046/j.1365-3148.1996.d01-518.xSearch in Google Scholar
28. Luo M, He J, Chen F. 54 cases of newborn multiple exchange transfusion resulting from severe ABO incompatibility. Chongqing Med J 2010:232–3 (in Chinese).Search in Google Scholar
29. Sherer DM, Abramowicz JS, Ryan RM, Sheils LA, Blumberg N, Woods JR Jr. Severe fetal hydrops resulting from ABO incompatibility. Obstet Gynecol 1991;78:897–902.Search in Google Scholar
30. Doyle B, Quigley J, Lambert M, Crumlish J, Walsh C, McParland P, et al. A correlation between severe haemolytic disease of the fetus and newborn and maternal ABO blood group. Transfus Med 2014;24:239–43.10.1111/tme.12132Search in Google Scholar PubMed
©2015 by De Gruyter
Articles in the same Issue
- Frontmatter
- Editorial
- Biomarkers of inflammatory bowel disease: ready for prime time?
- Review
- Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis
- Mini Reviews
- Cell-free DNA for diagnosing myocardial infarction: not ready for prime time
- The Laboratory Medicine and the care of patients infected by the Ebola virus. Experience in a reference hospital of Madrid, Spain
- Opinion Papers
- Theranos phenomenon − part 2
- Considerations in parathyroid hormone testing
- Quantity quotient reporting. Comparison of various models
- Genetics and Molecular Diagnostics
- Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks
- Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience
- Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR
- Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis
- General Clinical Chemistry and Laboratory Medicine
- European views on patients directly obtaining their laboratory test results
- Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients
- Thrombin generation, D-dimer and protein S in uncomplicated pregnancy
- Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry
- Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing
- Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease
- Reference Values and Biological Variations
- Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values
- Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV)
- Infectious Diseases
- Inosine triphosphate pyrophosphohydrolase activity: more accurate predictor for ribavirin-induced anemia in hepatitis C infected patients than ITPA genotype
- The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites
- Letter to the Editors
- Misleading high-sensitivity troponin algorithm for NSTEMI in the ESC guidelines
- Adjustment of serum potassium for age and platelet count. A simple step forward towards personalized medicine
- The impact of Tween 20 on repeatability of amyloid β and tau measurements in cerebrospinal fluid
- Effect of storage time and temperature on the generation of reactive oxygen species in peripheral blood leukocytes
- Iohexol interference in the α2-globulin fraction of the serum protein capillary electrophoresis
- A functional variant in the γ-glutamyltransferase (GGT)1 gene is associated with airflow obstruction in smokers
- Comparison of measured venous carbon dioxide and calculated arterial bicarbonates according to the PaCO2 and PaO2 cut-off values of obesity hypoventilation syndrome
- Creatinine, Jaffe, and glucose: another inconvenient truth
- Analytical evaluation of a new liquid immunoturbidimetric assay for the determination of ferritin in serum
- Analytical assessment of the novel homocysteine liquid enzymatic assay on Beckman Coulter AU5800
- Immunoglobulin IgA, IgD, IgG, IgM and IgG subclass reference values in adults
Articles in the same Issue
- Frontmatter
- Editorial
- Biomarkers of inflammatory bowel disease: ready for prime time?
- Review
- Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis
- Mini Reviews
- Cell-free DNA for diagnosing myocardial infarction: not ready for prime time
- The Laboratory Medicine and the care of patients infected by the Ebola virus. Experience in a reference hospital of Madrid, Spain
- Opinion Papers
- Theranos phenomenon − part 2
- Considerations in parathyroid hormone testing
- Quantity quotient reporting. Comparison of various models
- Genetics and Molecular Diagnostics
- Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks
- Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience
- Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR
- Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis
- General Clinical Chemistry and Laboratory Medicine
- European views on patients directly obtaining their laboratory test results
- Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients
- Thrombin generation, D-dimer and protein S in uncomplicated pregnancy
- Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry
- Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing
- Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease
- Reference Values and Biological Variations
- Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values
- Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV)
- Infectious Diseases
- Inosine triphosphate pyrophosphohydrolase activity: more accurate predictor for ribavirin-induced anemia in hepatitis C infected patients than ITPA genotype
- The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites
- Letter to the Editors
- Misleading high-sensitivity troponin algorithm for NSTEMI in the ESC guidelines
- Adjustment of serum potassium for age and platelet count. A simple step forward towards personalized medicine
- The impact of Tween 20 on repeatability of amyloid β and tau measurements in cerebrospinal fluid
- Effect of storage time and temperature on the generation of reactive oxygen species in peripheral blood leukocytes
- Iohexol interference in the α2-globulin fraction of the serum protein capillary electrophoresis
- A functional variant in the γ-glutamyltransferase (GGT)1 gene is associated with airflow obstruction in smokers
- Comparison of measured venous carbon dioxide and calculated arterial bicarbonates according to the PaCO2 and PaO2 cut-off values of obesity hypoventilation syndrome
- Creatinine, Jaffe, and glucose: another inconvenient truth
- Analytical evaluation of a new liquid immunoturbidimetric assay for the determination of ferritin in serum
- Analytical assessment of the novel homocysteine liquid enzymatic assay on Beckman Coulter AU5800
- Immunoglobulin IgA, IgD, IgG, IgM and IgG subclass reference values in adults
