Anti-platelet factor 4 (PF4) antibodies caused thrombocytopenia and thrombotic complications in COVID-19 vaccinees in rare cases, a syndrome known as vaccine-induced immune thrombotic thrombocytopenia (VITT). Anti-PF4 antibodies that caused VITT following Ad26.COV2.S vaccination was still unknown, and presented diagnostic techniques could not distinguish Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from related clinical diseases such as heparin-induced thrombocytopenia (HIT) and spontaneous HIT. While Ad26.COV2.S-associated VITT patients were invariably substantially positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they were usually negative in serotonin release assays (SRA). The PF4-dependent p-selectin expression assay (PEA) used platelets treated with PF4 instead of heparin to detect Ad26.COV2.S-associated VITT was found to be accurate. In PF4-polyanion ELISAs, most Ad26.COV2.S-associated VITT antibodies lasted for more than 5 months, whereas the PEA became negative quicker. After 6 months following acute presentation, 2 patients developed mild persistent thrombocytopenia (140-150 x 103/µL), otherwise unexplained. Differentiating VITT from spontaneous HIT originated in the absence of local heparin exposure. HIT was crucial from an epidemiological standpoint; however, available PF4-polyanion ELISAs and functional assays were non-specific and detected in all 3 situations. Researchers showed that a novel uncomplicated PF4 ELISA could distinguish VITT, which was caused by both Ad26.COV2.S and ChAdOx1 nCoV-19 from spontaneous HIT and HIT-suspected patients were PF4/polyanion ELISA-positive but negative in functional assays. In conclusion, Ad26.COV2.S-associated VITT antibodies were persistent, and the uncomplexed PF4 ELISA for VITT diagnosis appeared to be both sensitive and specific.