Abstract
Background - Non-invasive fetal HPA typing is a valuable tool to identify the pregnancies at risk of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Different approaches have been developed, mainly based on real-time PCR and droplet digital-PCR. Those methods have a limited ability to multiplex and require replicates due to the contamination risk. Moreover, in order to exclude false-negative results caused by insufficient cell-free fetal DNA, the presence of fetal DNA is usually assessed with a single epigenetic marker whereas large single-nucleotide variant (SNV) panels are now available to more accurately measure the sample’s fetal fraction.
Materials and methods - We developed an innovative method for the simultaneous genotyping of HPA-1, -2, -3, -4, -5, -6, -9 and 15, in combination with a panel of 48 SNV markers, based on cell-free DNA target-enrichment with specific probes. An improved accuracy using NGS sequencing was reached with the Unique Molecular Identifiers (UMIs); these molecular barcodes are short sequences used to uniquely tag each molecule in a sample library.
Results - 81 plasma samples were collected from French and Spanish pregnant women, from 10 to 40 weeks of gestation ([wg]; 4 samples <12 wg; 38 samples 13-24 wg; 39 samples >24 wg). The panel of 48 SNVs allowed a precise quantification of fetal DNA (range: 1.1%-16.1%). All samples but one gave concordant results with the confirmed HPA genotypes. One sample was discordant for HPA-2 and HPA-3, due to false negative cffDNA results for these two loci. However, a low amount of fetal fraction in that sample was effectively alerted by the SNV markers results.
Discussion - In our experience, 16 samples can be simultaneously sequenced and analyzed in a 72 hours assay. UMIs NGS sequencing of HPA and SNV markers constitutes a robust and sensitive method for non-invasive fetal HPA genotyping.
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Authors
Gerald Bertrand
EFS BloodCenter of Brittany, HLA-HPA Laboratory, Rennes, France
Orlane Levallois EFS BloodCenter of Brittany, HLA-HPA Laboratory, Rennes, France
Cecilia Gonzalez Santesteban
Immunohematology Laboratory, Banc de Sang iTeixits, Barcelona, Spain
Nuria Nogues
Immunohematology Laboratory, Banc de Sang iTeixits, Barcelona, Spain
Virginie Renac EFS BloodCenter of Brittany, HLA-HPA Laboratory, Rennes, France
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