Fetal and neonatal alloimmune thrombocytopenia: Current pathophysiological insights and perspectives for future diagnostics and treatment

Stam, Wendy; Wachholz, Gabriela Elis; Pereda, José M. de; Kapur, Rick; Schoot, Ellen van der; Margadant, Coert

doi:10.1016/j.blre.2022.101038

Cited by 9 publications

(8 citation statements)

References 91 publications

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“…The pathophysiology of FNAIT is complex and involves multiple possible mechanisms ( 40 ) that, in addition to causing platelet destruction through IgG-Fc receptors, may also be affected by complement activation ( 41 ) and CRP ( 42 ). Platelet production can also be affected by the immune response targeting megakaryocytes ( 43 ), and the antibodies can also cause vascular damage by binding to HPA antigens, which are also expressed on endothelial cells ( 44 ) and trophoblast cells ( 45 ).…”

Section: Discussionmentioning

confidence: 99%

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia

Szittner,

Bentlage,

Temming

et al. 2023

Front. Immunol.

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Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

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Section: Discussionmentioning

confidence: 99%

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia

Szittner,

Bentlage,

Temming

et al. 2023

Front. Immunol.

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“… 1 , 35 In addition, antibodies can have functional effects on integrins (some of which related to the stabilization or alteration of specific integrin conformations) and this can affect integrin localization and behavior. 34 , 37 , 38 Transfer 1 or 2 coverslips directly after labeling to a well containing 4% PFA. These coverslips will reveal integrin distribution at the cell-surface before the onset of internalization (t = 0) ( Figure 9 ).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Imaging and quantitative analysis of integrin-dependent cell-matrix adhesions

van Stalborch,

Clark,

Sonnenberg

et al. 2023

STAR Protocols

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“…5,6 The clinical presentation of FNAIT varies, encompassing a wide spectrum of symptoms ranging from no bleeding to severe ICH and intrauterine fetal death. 7 Over the last years, evidence has accumulated that the specificity and functional characteristics of HPA-1aspecific antibodies are heterogeneous [8][9][10][11][12][13][14] and might be linked to disease outcomes. 15,16 Although all antibodies target the HPA-1a epitope, formed by a single amino acid substitution (Leu33Pro) on integrin β3, their specific footprint varies and differentially affects target cells.…”

Section: Introductionmentioning

confidence: 99%

Generation of human antibodies targeting human platelet antigen (HPA)‐1a

Oosterhoff,

Linty,

Visser

et al. 2024

Transfusion

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BackgroundFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)‐1a accounts for the majority of FNAIT cases. Binding of HPA‐1a‐specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA‐1a‐specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease.Study Design and MethodsThis study aimed to isolate HPA‐1a‐specific B‐cells from an HPA‐1a‐alloimmunized pregnant woman. Using fluorescently labeled HPA‐1a‐positive platelets, single B‐cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme‐linked immunosorbent assay and their reactivity towards HPA‐1a‐positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK‐Freestyle‐based expression system.ResultsThree platelet‐specific B‐cells were obtained and cloned of which two were specific for HPA‐1a, named D‐ and M‐204, while the third was specific for HLA class I, which was named L‐204.DiscussionThis study outlined an effective method for the isolation of HPA‐1a‐specific B‐cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

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Fetal and neonatal alloimmune thrombocytopenia: Current pathophysiological insights and perspectives for future diagnostics and treatment

Cited by 9 publications

References 91 publications

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia

Imaging and quantitative analysis of integrin-dependent cell-matrix adhesions

Generation of human antibodies targeting human platelet antigen (HPA)‐1a

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