Supplementary Material for: IgG-Fc-glycosylation and a novel flowcytometric assay to predict Hemolytic Disease of the Fetus and Newborn

Introduction: Timely and accurate referral of pregnancies with red blood cell (RBC) alloantibodies is essential to prevent perinatal death or long-term adverse consequences in hemolytic disease of the fetus and newborn (HDFN). Classically, antibody titers are used to select high-risk pregnancies. Research suggested that also IgG-Fc-fucosylation could be an important pathological feature. We evaluated the diagnostic potential of IgG-Fc-fucosylation in identifying high-risk pregnancies, and we report on a flowcytometric assay that integrates antibody quantity, antibody subclass and fetal FcγRIIIa affinity.

Methods: Maternal serum samples from a nationwide prospective cohort of D-alloimmunized pregnancies, between 2014 and 2017, were used to evaluate IgG-Fc-glycosylation in mass-spectrometry (n=64), and the flowcytometric assay (n=90). Results were compared to standard-of-care titers and antibody-dependent cellular-cytotoxicity assay (ADCC) results. Receiver operating curves (ROC) were used to assess and quantify predictive values.

Results: The cohort for mass-spectrometry analysis consisted of 31 pregnancies without transfusions (48%), 16 with IUT (25%) and 17 (27%) with postnatal transfusions only. The flowcytometric assay consisted of 53 (59%) pregnancies without transfusions, 17 (19%) pregnancies with IUT, and 20 (22%) pregnancies with postnatal transfusions only, suggesting that severe HDFN was overrepresented. Anti-D IgG1 and IgG3-Fc-fucosylation levels were lower in groups requiring transfusions, but no distinct difference between groups was detected. No significant difference in other glycan traits was found. Among sera from 90 D-alloimmunized pregnancies evaluated in the flowcytometric assay, we found significant differences in IgG- and FcγRIIIa binding between pregnancies without transfusions and with IUT, suggesting a potential predictive value for the need for IUTs. ROC analyses revealed a lower false-positive rate at a 100% sensitivity in the prediction of pregnancies at-risk for fetal anemia through the flowcytometric assay (20.8% for IgG binding and 43.1% for FcγRIIIa binding) compared to the titer and ADCC (74.6%).

Conclusion: Anti-D IgG-Fc-fucosylation levels tended to be lower in pregnancies with IUT, but could not distinguish pregnancies with or without IUT in this cohort. The flowcytometric assay, measuring IgG levels and functional FcγRIIIa binding, indicates that the specificity of serological monitoring in HDFN may be improved. Considering an overrepresentation of severe HDFN in this cohort, we advise to confirm these findings in a prospective cohort within a routine setting.