DETERMINATION OF FETAL RHESUS D STATUS BY MATERNAL PLASMA DNA ANALYSIS
Aykut A1,*, Onay H1, Sagol S2, Gunduz C3, Ozkinay F1, Cogulu O1
*Corresponding Author: Ayça Aykut, M.D., Ph.D., Department of Medical Genetics, Ege University Faculty of Medicine, 35100, Bornova, Izmir, Turkey; Tel.: +90-232-390-3961; Fax: +90-232-390-3971; E-mail: aycaaykut@hotmail.com
page: 33

MATERIALS AND METHODS

Blood samples (9 mL), collected in EDTA vacutainers, from 30 RhD-negative Turkish women between 9 and 39 weeks of gestation, who were referred to us for invasive testing because of advanced maternal age, increased maternal serum screening test, fetal sonographic abnormality and previous history of chromosomal or single gene disorder. Routine assay for ABO and RhD typing and testing for unexpected antibodies were performed to include RhD negative women in the study. The positive control for the RHD and SRY genes was a heterozygous RHD-positive man, while the negative control for both genes was an RhD-negative non pregnant woman. The SRY gene served as an internal control marker to confirm the presence of male fetal DNA. All analyses were performed blind, that is, the fetal RHD genotyping was performed without knowing the fetus RhD status, which was confirmed by serological methods postpartum. Nine mL of maternal blood was collected in EDTA vacutainers and sent to the laboratory at room temperature. The blood was centrifuged at 2840 rpm for 10 min., the plasma was transferred without disturbing the buffy coat and recentrifuged again at 3600 rpm for 20 min. and the supernatants were collected and stored at −80 °C before DNA extraction. Written informed consent was obtained from all the families. The study was approved by the Faculty Ethics Committee of Ege University Faculty of Medicine, Izmir, Turkey. DNA Extraction from Plasma Samples and Fetal Samples. DNA was extracted from 500 mL plasma using QIAamp DSP Virus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was eluted in 20 Elution buffer (AVE) and 4.0 μL was used as a template for the polymerase chain reaction (PCR). DNA from amniocentesis or CVS specimens was isolated using Chelex (InstaGene Matrix™, Bio-Rad Laboratories, Mississauga, Ontario, Canada) in a rapid isolation technique according to the manufacturer’s instructions. The specimens were stored at −20 °C before being studied. Real-time Polymerase Chain Reaction Analysis. The TaqMan real-time PCR assay protocol (LightCycler 1.5, Roche Diagnostics, Mannheim, Germany) was performed. The primers and probes used for RHD genotyping were targeted towards exons 7 and 10. For the detection of chromosome Y, primers and probes were targeted for the SRY gene on chromosome Y (Table 1). Amplicon lengths for exons 7, 10 and SRY were 82, 122 and 137 bp, respectively. All primers and probes were synthesized by TIB MOLBIOL (Berlin, Germany). At least two regions of the RHD gene were used for the complex genetic variant forms of RHD. The primers and probes designed for exons 7 and 10 did not permit amplification of a non functional rearranged RHD gene, which has been demonstrated in this and in other studies generating positive results in exon 10. The RHD/SRY amplification reactions were set up in a volume of 20 mL. Each reaction contained 4 mL of Light Cycler DNA Master Hybridization Probes (Roche Diagnostics, Basel, Switzerland; 10× concentrated), 100 nM of each probe, and 200 nM of each amplification primer. A 4 mL volume of the extracted DNA was used for amplification. Thermal cycling was initiated by a denaturation step of 10 min. at 95 °C, followed by 50 cycles at 95 °C for 15 seconds and at 60 °C for 60 seconds. Amplification data were analyzed using the Light-Cycler software (Roche Diagnostics). A duplicated RHD/SRY PCR was set up for each individual sample. If results in the duplicate tests were inconclusive or all data for the RHD and SRY genes were negative, the PCR set up was repeated, resulting in a maximum number of four RHD/SRY PCR procedures. The presence or absence of the fetal RHD gene was determined as follows: first, the samples were examined in duplicate. If both reactions were positive, we considered the fetus was carrying the paternal RHD gene. If both reactions were negative or one of two was positive, the sample was repeated in another duplicate. In total, if the fetal RHD sequence was detectable in two or more of four reactions, the result was scored as positive. Otherwise, it was considered to be negative. Confirmation of our results was performed by the analysis of the serological test on cord blood after delivery or by genotyping the fetal DNA obtained from amniocentesis or CVS. Only samples with cycle threshold (Ct) values of less than 40 were considered positive. The results were finally interpreted if all performed PCR procedures for RHD and SRY were consistent. If the PCR replicates for RHD or SRY were discrepant, the plasma was retested from DNA isolated from the second tube. For gender detection, the SRY sequence was used to identify male fetal DNA that was present in each sample. The experiment was repeated in three separate reactions. If at least two of the three amplification reactions were positive for the SRY sequence, the fetus was considered to be male. If there was no SRY sequence amplification in all separate reactions, the fetus was considered to be female. The results were confirmed by karyotype, examination after birth or Doppler ultrasound after 20 weeks of gestation.



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