Developing a flow cytometry detection method for detecting alpha V beta 3 antibodies which have been implicated in neonatal alloimmune thrombocytopenia cases (NAIT)

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.