Prediction of severity of neonatal alloimmune thrombocytopenia (NAIT) through risk stratification by assessing the IgG subclasses involved

Abstract

NAIT can be a fatal condition that affects the fetus or new-born. Maternally derived IgG alloantibodies directed against paternally inherited specific platelet antigens (HPA) cross the placenta and target fetal platelets. The most severe cases result in death due to intra-cranial haemorrhage (ICH). Reports suggest that a compound antibody to integrin alpha V beta 3 is also present in maternal samples positive for antibodies against HPA-1a. Alpha V beta 3 is required for angiogenesis therefore ICH could be due to impaired endothelial development caused by function-blocking antibodies to anti-alpha V beta 3.

This study aimed to develop an assay that could detect alpha V beta 3 antibodies. Human endothelial kidney (HEK) cells transfected with an expression vector forming the heterodimers alpha V and beta 3 or glycoproteins IIb/IIIa (alpha 2a beta 3) were created. These were validated using monoclonal antibodies and known HPA positive sera by flow cytometry and anti-mouse/human conjugate. Transfected cell clones were incubated with serum samples (n=24) from cases referred for NAIT testing positive for HPA-1a antibodies. Analysis using flow cytometry revealed that all samples reacted with both HEK clones; cut-off established using 4SDs above the mean of 6 negative control sera. Dilution of samples revealed that 3/5 ICH samples had increased positive reactions with the alpha V beta 3 protein, compared to 1 non-ICH sample. When testing with IIb/IIIa transfected cells, one ICH sample had an increased positive ratio on dilution. This indicates that the titre to the compound HPA-1a antibody is stronger in ICH cases. In conclusion, an assay has been produced that detects alpha v beta 3 antibodies; these antibodies cannot be differentiated from IIb/IIIa antibodies without future adsorption studies. Both HPA-1a and HPA-1b versions of beta 3 for alpha V and IIb proteins were produced and possessed relevant epitopes, these have potential antibody screening applications. Studies on kidney transplantation have indicated that donor specific HLA antibodies of the IgG4 subtype can predict rejection.

This study investigated if IgG subtype analysis could be used to predict ICH in known NAIT cases. Forty samples (20 with and 20 without ICH) were selected from NAIT cases with HPA-1a antibodies detected by the monoclonal antibody immobilisation of platelet antigens (MAIPA). Previous studies using MAIPA have indicated that IgG1 and IgG3 isotypes are present in NAIT but not linked to severity. Here, Luminex technology using an in-house multiplex bead assay with recombinant HPA-1a, HPA-1b and GPVI proteins was tested. Cut-offs were established using One Lambda’s normalised background ratio plus 4SDs from 22 female apheresis donors. Each sample was tested for total IgG and a separate phycoerythrin conjugate for each IgG isotype. Seven samples did not react with the recombinant proteins; two samples were positive for total IgG but negative for all isotypes. Fifteen cases with ICH were positive for total IgG; of these all were positive for IgG1, 5 for IgG2, 5, for IgG3 and 4 for IgG4. Sixteen non-ICH samples were positive for total IgG and 14, 5, 8 and 3 were positive for IgG1, IgG2, IgG3 and IgG4 respectively. This study indicates that there is no difference in IgG isotypes between ICH and non-ICH NAIT cases. Further statistical analysis will analyse ratios with outcomes and compare with different cohorts such as HPA refractory and post transfusion purpura patients.

Outputs

No changes in practice as of yet. Submitted two abstracts to BSHI/EFI Conference which has been postponed until 2021.