RHCE*E positive droplets were detected for a 10 week GA case with maternal anti-E tested in 5 replicates, average RHCE*E concentration 0.5 copies/μL predicting an E positive fetus (H02-D03). QuantaSoft™ fluorescence amplitude plots showing detection of fetal blood group alleles in maternal cfDNA in singleplex and duplex ddPCR assays for two cases: (A) is a one dimensional (1D) plot for NIPT RHCE*E fetal signals No template control (NTC) and genomic DNA controls were included on the plate (F04-D02). Probe sequence, labels (5′–3′) and manufacturerĥ6-FAM/TTT AAC CGA/ZEN/A TG CTG AGA CT/3IABkFQ *ĥHEX/TTT AAC CGA/ZEN/A CG CTG AGA CT/3IABkFQ *ĥ6-FAM/CCA GGT TGG/ZEN/CA C CAT AGT CTC CA/3IABkFQ *ĥHEX/CCA GGT TGG/ZEN/CA T CAT AGT CTC CA/3IABkFQ *ĦFAM/CAA TCC TGC TGG ACG GCT TCC TGA/TAMRA †ĦFAM/CCA AAG GAA GAA TGC CAT GTT CAA CAC CTA C/TAMRA †ĦFAM/TTT CCT TCT TAC TGT CCC CTT CTG GGC TC/TAMRA †ĥHEX/ACA GCG ATG/ ZEN/ATT ACA GTC CAG C/3IABkFQ *Įxon 1, ACTB 5′UTR and exon 1 (Chan et al., 2006) All assays performed optimally under the same thermal cycling conditions permitting a simplified laboratory protocol, Table 1. For the singleplex assays, a separate housekeeper DNA control, CCR5, was included, measuring total plasma (maternal and fetal) cfDNA levels (Finning et al., 2002). For the duplex assays, the maternal allele serves both as a ‘housekeeper’ DNA control marker and allows measurement of the fetal fraction of the cfDNA component. The NIPT suite comprised duplex assays for KEL*01.01/KEL*02 (K/k) and FY*A/FY*B (Fy a/Fy b), and published singleplex assays for RHCE*c (c), RHCE*E (E) (Finning et al., 2007) and RHCE*C (C) (Legler et al., 2002). Each droplet is expected to contain zero or one copy of either maternal or fetal cell-free (cf) DNA, removing the competition effects caused by mcfDNA sequences. The ddPCR partitions each reaction mixture into tens of thousands of droplets or ‘nano-reactions’ prior to amplification. To manage the challenge of detecting low copy number fetal alleles in complex samples, we evaluated a droplet digital PCR (ddPCR) approach for a suite of non-RhD NIPT blood group assays. This places restrictions on the use of real-time PCR, specifically in the case of K (Finning et al., 2007 Scheffer et al., 2011). ![]() Adverse pre-analytic sample conditions which result in maternal white cell lysis further increase background mcfDNA and reduce the fraction of cffDNA, which itself is in low abundance (Lun et al., 2008). For this reason, there are limitations for NIPT using real-time PCR assays due to competition from background maternal cell-free (mcf) allelic DNA sequences. In contrast to RhD, the majority of blood group antigens, including K, c, E, C and Fy a/Fy b, arise from single nucleotide variant(s) (SNVs). ![]() There are therefore no maternal background DNA sequences to compete for detection with fetal RHD sequences. Conventional real-time PCR allows ready detection of fetal RHD sequences because the RHD gene is deleted on the D-negative haplotype in the majority of cases (Colin et al., 1991). NIPT has proven reliable for fetal RHD genotyping for D-negative pregnant women (Finning et al., 2002 Mackie et al., 2016 Hyland et al., 2017). Non-invasive prenatal testing (NIPT) of cell-free fetal (cff) DNA in maternal plasma determines whether the target paternal blood group allele has been inherited, thus predicting whether the fetus is at risk for HDFN. ![]() For anti-D, anti-K, and anti-c, there is a high (>50%) risk for mild to severe haemolytic disease of the fetus or newborn (HDFN) developing should the fetus inherit the target paternal red cell antigen (de Haas et al., 2015). The most clinically significant antibodies are anti-D (in the Rh blood group system) and anti-K (Kell system), followed by anti-c, anti-E, anti-C (Rh system), and anti-Fy a or anti-Fy b (Duffy system) (Royal College of Obstetricians and Gynaecologists, 2014). Pregnant women who present with an antibody to a blood group antigen may require intensive monitoring throughout pregnancy to manage early warning signs of fetal anemia.
0 Comments
Leave a Reply. |