Investigation of anti-PF4 versus anti-PF4/heparin reactivity using fluid-phase enzyme immunoassay for 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia (HIT), autoimmune HIT, vaccine-induced immune thrombotic thrombocytopenia, and spontaneous HIT

Four platelet-activating anti-platelet factor 4 (PF4) disorders have been recognized: classic heparin-induced thrombocytopenia (cHIT), autoimmune heparin-induced thrombocytopenia (aHIT), spontaneous heparin-induced thrombocytopenia (SpHIT), and vaccine-induced immune thrombotic thrombocytopenia (VITT). All test immunoglobulin G (IgG) positive using solid-phase enzyme immunoassay (solid-EIA) against PF4/heparin (PF4/H) and/or PF4 alone. Fluid-phase EIA (fluid-EIA) should better discriminate between anti-PF4 and anti-PF4/H antibodies since conformationally altered PF4 bound to solid phase is avoided. To compare anti-PF4 vs anti-PF4/H antibody profiles for anti-PF4 disorders using solid- and fluid-EIA. We developed a novel fluid-EIA to measure anti-PF4 vs anti-PF4/H antibodies. Using fluid-EIA, 27 of 27 (100%) cHIT sera tested IgG positive with PF4/H, but only 4 of 27 (14.8%) tested positive against PF4 alone; all 27 exhibited heparin-enhanced binding. In contrast, 17 of 17 (100%) VITT sera tested IgG positive against PF4 alone, with markedly reduced binding against PF4/H; this distinct VITT antibody profile was not evident using solid-EIA. All 15 aHIT sera and all 11 SpHIT sera tested IgG positive against PF4 alone, with variable reactivity in PF4/H-EIA (heparin-enhanced binding in 14 of 15 and 10 of 11 aHIT and SpHIT sera, respectively). Remarkably, 1 SpHIT patient with a VITT-mimicking fluid-EIA profile (PF4 >> PF4/H) also clinically resembled patients with VITT (postviral cerebral vein/sinus thrombosis), with anti-PF4 reactivity correlating inversely with platelet count recovery; moreover, the single aHIT patient with a VITT-mimicking fluid-EIA profile also developed postviral cerebral vein/sinus thrombosis. cHIT and VITT sera showed opposite fluid-EIA profiles (cHIT: PF4/H >> PF4, with most testing negative against PF4 alone; VITT: PF4 >> PF4/H, with most testing negative against PF4/H). In contrast, all aHIT and SpHIT sera reacted against PF4 alone but with variable (usually enhanced) reactivity against PF4/H. VITT-mimicking clinical/serologic profiles occurred in only a minority of patients with SpHIT and aHIT. •Fluid-phase enzyme immunoassay (EIA)—vs solid-phase EIA—avoids conformational alteration of platelet factor 4 (PF4).•Using fluid-phase EIA, classic heparin-induced thrombocytopenia (HIT) sera recognize PF4/heparin (PF4/H) but usually not PF4 alone.•In contrast, autoimmune HIT, spontaneous heparin-induced thrombocytopenia (SpHIT), and vaccine-in