Non-invasive genotyping fetal Kell blood group (KEL1) using cell-free fetal DNA in maternal plasma by MALDI-TOF mass spectrometry. Y. Li¹, K. Finning², G. Daniels², W. Holzgreve¹, S. Hahn¹. 1) Department of Research, University of Basel, Basel, Switzerland; 2) International Blood Group Reference Laboratory, National Blood Service, Bristol, UK.
   Alloimmunization against the fetal Kell (KEL1) blood group antigen is gaining in importance and is the second most important cause of haemolytic disease of the newborn (HDN). The KEL*1 gene differs from the highly prevalent KEL*2 allele by a C-to-T base substitution in exon 6, thereby encoding threonine instead of methionine at residue 193. Currently, prenatal diagnosis of KEL1 involves invasive procedures, such as amniocentesis and chorionic villus sampling, which present a risk for both fetus and mother. The discovery of fetal cell-free DNA (cf-DNA) in maternal plasma provides the possibility for non-invasive prenatal diagnosis. However, the detection of fetal gene point mutations is difficult when using conventional PCR-based methods, due to the overwhelming presence of maternal cf- DNA sequences. In this context, it has recently been shown that the MALDI-TOF mass spectrometry-based single allele base extension reaction (SABER) assay may permit the detection of paternally inherited fetal SNPs from cf-DNA in maternal plasma. In this study, we examined KEL*1 from KEL:-1,2 pregnant women using the MALDI-TOF MS-based SABER assay. Thirty maternal plasma samples at risk for KEL1-alloimmunization were taken at 14-35 (mean: 22.8) weeks of gestation. The results were confirmed by serological tests on cord blood or PCR typing on amniocyte-derived fetal DNA. We detected the fetal KEL*1 allele in 11 of the 13 KEL1-positive samples (85% sensitivity). In total, the presence or absence of the paternal KEL*1 allele could be correctly determined in 93% of cases (28/30). Therefore, the MALDI-TOF mass spectrometry-based SABER assay may be useful for the detection of the fetal KEL1 status, but needs to be improved for clinical application. More precise methods, such as size fractionation of cf-DNA approach, might help improve the detection.