Differentiating pathogenic from bystander autoantibodies in immune thrombocytopenia using intact glycoprotein-deficient megakaryocytes

Autoantibodies targeting platelet surface glycoproteins (GP) are the primary cause of immune thrombocytopenia (ITP), however testing for these autoantibodies is not routinely employed mainly because current diagnostic tests lack sufficient sensitivity. To overcome this and other diagnostic limitations, we generated HLA Class I-negative, blood group O, induced pluripotent stem cell lines, and gene-edited them to produce a novel panel of GP-deficient, in vitro-derived megakaryocytes (MK). Using GPIIb-, GPIbα- and GPIX-deficient MK allowed sensitive and specific identification and characterization of both GPIIb-IIIa- and GPIb/IX-specific plasma autoantibodies, as well as rare, previously undescribed patient autoantibodies targeting GPIX. The availability of frozen bioengineered MK expressing select platelet glycoprotein antigens on their surface simplifies the detection of anti-platelet autoantibodies involved in disease progression, while avoiding detection of non-pathogenic bystander autoantibodies that are sometimes generated by secondary exposure to “cryptic epitopes”, and that can otherwise confound diagnosis and treatment. Use of intact MK selectively deficient in the glycoproteins that comprise the major targets of platelet autoantibodies has the potential to significantly improve clinical diagnosis and treatment of ITP.