Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing.

BACKGROUND: Fetal human platelet antigen (HPA) genotyping is required to determine whether the fetus is at risk and whether prenatal interventions to prevent fetal bleeding are required in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Methods for noninvasive genotyping of HPA alleles with the use of maternal plasma cell-free DNA were published recently but do lack internal controls to exclude false-negative results. STUDY DESIGN AND METHODS: Cell-free DNA was isolated from plasma of four pregnant women with a history of FNAIT caused by anti-HPA-1a and controls. A primer panel was designed to target sequences flanking single-nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA-1), ITGA2B (HPA-3), ITGA2 (HPA-5), CD109 (HPA-15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and eight anonymous SNPs were massively parallel sequenced by semiconductor technology. RESULTS: The mean (±SD) number of reads for targeted SNPs was 5255 (±2838). Fetal DNA was detected at a median of 4.5 (range, 2-8) polymorphic loci. The mean fractional fetal DNA concentration in cell-free maternal plasma was 8.36% (range, 4.79%-15.9%). For HPA-1, nonmaternal ITGB3 sequences (c.176T, HPA-1a) were detected in all HPA-1ab fetuses. One HPA-1bb fetus was unequivocally identified, showing the pregnancy was not at risk of FNAIT. CONCLUSION: We have successfully established massively parallel sequencing as a novel reliable method for noninvasive genotyping of fetal HPA-1a alleles. This technique may also allow the safe detection of other fetal blood group polymorphisms frequently involved in FNAIT and hemolytic disease of the newborn.

Glycosylation of autoantibodies: insights into the mechanisms of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets (PLTs). IgG effector functions depend on their Fc-constant region which undergoes posttranslational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. AAbs were purified from ITP patients (n=15) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and activated complement. N-glycan cleavage significantly reduced both effects. In vivo survival of human PLTs was assessed after co-transfusion with native or N-glycan cleaved AAbs in a NOD/SCID mouse model. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL(5h))75% vs 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL(5h) 82% vs 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL(5h) 42%, p<0.001). N-glycosylation of human ITP AAbs appears to be required for platelet phagocytosis and complement activation, reducing platelet survival in vivo. Posttranslational modification of AAbs may constitute an important determinant for the clinical manifestation of ITP.

Inhibition of HPA-1a alloantibody-mediated platelet destruction by a deglycosylated anti-HPA-1a monoclonal antibody in mice: toward targeted treatment of fetal-alloimmune thrombocytopenia.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is often caused by maternal alloantibodies against the human platelet antigen (HPA)-1a, which opsonizes fetal platelets (PLTs). Subsequent PLT destruction is mediated via the Fc part of the alloantibodies. The monoclonal antibody (mAb) SZ21 binds to the HPA-1a epitope and inhibits the binding of maternal alloantibodies. However, it also promotes complement activation and phagocytosis. Deglycosylation of antibodies abrogates the Fc-related effector functions. We modified the N-glycan of SZ21 by endoglycosidase F. The in vivo transplacental transport of N-glycan-modified (NGM)-SZ21 was not impaired. When injected into pregnant mice, both native-SZ21 and NGM-SZ21 were transported equally into fetal circulation (8.9% vs 8.7%, respectively, P = .58). Neither the binding properties of NGM-SZ21 to HPA-1a in surface plasmon resonance, nor the inhibition of anti-HPA-1a-induced PLT phagocytosis, were affected by N-glycan modification. NGM-SZ21 prevented PLT destruction induced by maternal anti-HPA-1a antibodies in vivo in a mouse model (PLT clearance after 5 hours; 18% vs 62%, in the presence or absence of NGM-SZ21, respectively, P = .013). Deglycosylation of SZ21 abrogates Fc-effector functions without interfering with placental transport or the ability to block anti-HPA-1a binding. Humanized, deglycosylated anti-HPA-1a mAbs may represent a novel treatment strategy to prevent anti-HPA-1a-mediated PLT destruction in FNAIT.

The implementation of surface plasmon resonance technique in monitoring pregnancies with expected fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Maternal anti-HPA-1a alloantibodies are responsible for most cases of severe fetal and neonatal alloimmune thrombocytopenia (FNAIT). The presence of HPA-1a alloantibodies in maternal blood alone does not predict the fetal platelet (PLT) count, and the predictivity of antibody titers determined by enzyme immunoassays (EIAs) is debated. In contrast to EIA, surface plasmon resonance (SPR) provides information on antibody-binding properties. STUDY DESIGN AND METHODS: Sequential sera from pregnant women with expected FNAIT were assessed for HPA-1a alloantibodies using SPR. Group I (n = 6) was treated with intravenous immunoglobulin (IVIG) and steroids beginning at 19 weeks of gestation (w.g.), and Group II (n = 4) received intrauterine PLT transfusions (IUT) beginning at 22 w.g. Maternal alloantibodies were quantified using an HPA-1a monoclonal antibody (MoAb) as a standard. Antibody avidity was determined as the ratio of B700 (end of the dissociation phase) to B350 (end of the association phase); the area under the curve (AUC) was calculated to determine overall antibody binding. RESULTS: After 22 w.g., alloantibody characteristics remained stable in both groups, while there was a steep decrease in B700 and B350 values between 16 and 22 w.g. (assessed only in Group I), indicating a decrease in anti-HPA-1a alloantibody concentrations. Interestingly, the AUCs of the last maternal sample before elective delivery appeared to be correlated with fetal and neonatal PLT counts (p = 0.014 and 0.017, respectively). CONCLUSION: SPR provides quantitative information on HPA-1a alloantibody characteristics in addition to monoclonal antibody-specific immobilization of platelet antigens. SPR results can be calibrated using a MoAb standard and should be further assessed for a potential correlation with fetal PLT count.

Low-avidity anti-HPA-1a alloantibodies are capable of antigen-positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA-1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti-HPA-1a could be overlooked by standard methods due to low avidity. Low-avidity HPA-1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low-avidity anti-HPA-1a. STUDY DESIGN AND METHODS: A retrospective cohort consisting of 82 HPA-1bb mothers of HPA-1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low-avidity antibodies in SPR using purified GPIIb/IIIa (HPA-1a or -1b). The capability of HPA-1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. RESULTS: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA-1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA-negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA-1a (mean, 31 ± 5 RU), but not to HPA-1b flow cell (mean, 5 ± 2 RU). In vivo, low-avidity antibodies were capable of clearing HPA-1ab PLTs but not HPA-1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA-positive antibodies. CONCLUSIONS: Low-avidity HPA-1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.