Persistence of Ad26.COV2.S-associated vaccine-induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies

Abstract

Rare cases of COVID-19 vaccinated individuals develop anti-platelet factor 4 (PF4) antibodies that cause thrombocytopenia and thrombotic complications, a syndrome referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). Currently, information on the characteristics and persistence of anti-PF4 antibodies that cause VITT after Ad26.COV2.S vaccination is limited, and available diagnostic assays fail to differentiate Ad26.COV2.S and ChAdOx1 nCoV-19-associated VITT from similar clinical disorders, namely heparin-induced thrombocytopenia (HIT) and spontaneous HIT. Here we demonstrate that while Ad26.COV2.S-associated VITT patients are uniformly strongly positive in PF4-polyanion enzyme-linked immunosorbent assays (ELISAs); they are frequently negative in the serotonin release assay (SRA). The PF4-dependent p-selectin expression assay (PEA) that uses platelets treated with PF4 rather than heparin consistently diagnosed Ad26.COV2.S-associated VITT. Most Ad26.COV2.S-associated VITT antibodies persisted for >5 months in PF4-polyanion ELISAs, while the PEA became negative earlier. Two patients had otherwise unexplained mild persistent thrombocytopenia (140-150 x 10³ /µL) 6 months after acute presentation. From an epidemiological perspective, differentiating VITT from spontaneous HIT, another entity that develops in the absence of proximate heparin exposure, and HIT is important, but currently available PF4-polyanion ELISAs and functional assay are non-specific and detect all three conditions. Here, we report that a novel un-complexed PF4 ELISA specifically differentiates VITT, secondary to both Ad26.COV2.S and ChAdOx1 nCoV-19, from both spontaneous HIT, HIT and commonly-encountered HIT-suspected patients who are PF4/polyanion ELISA-positive but negative in functional assays. In summary, Ad26.COV2.S-associated VITT antibodies are persistent, and the un-complexed PF4 ELISA appears to be both sensitive and specific for VITT diagnosis.

FIGURE 1

VITT antibodies are strongly ELISA positive, activate PF4‐treated platelets, and are persistent. (A) PF4‐Polyanion ELISA testing from nine patients upon initial presentation with VITT is presented. The Y‐axis denotes Optical density, OD. Eight patients were tested in the LIFECODES PF4 IgG (IgG‐specific), while one was tested in the LIFECODES PF4 Enhanced assay (detects IgG, IgA, and IgM), both FDA‐approved in vitro diagnostic assays for HIT. Mean and standard error are shown. The dotted line represents the positive cut‐off of the assay. (B) SRA and PEA results upon initial presentation of six VITT patients are presented. Open circles, SRA; Closed circles, PEA. Mean and standard error are shown. The solid and dotted line represents the positive cut‐offs of the SRA and PEA, respectively. (C) Kaplan Meier curves for time to negative ELISA OD (<0.4 OD; yellow) and PEA (<19%; blue) are shown. Due to the small number of patients (n = 9), confidence intervals were wide (not shown). (D) Kaplan Meier curves for time to normalization of D‐dimer (cut‐off varied by testing laboratory, as shown in Table 1; blue) and platelets count (>150 x 10³/µL; yellow) are presented. Due to the small number of patients (n = 9), confidence intervals were wide (not shown) [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

PF4/polyanion ELISAs are sensitive but highly non‐specific, while an un‐complexed PF4 ELISA is both sensitive and specific for the detection of VITT antibodies. Binding of antibodies from five Ad26.COV2.S‐associated VITT (closed black circles), one ChAdOx1 nCoV‐19‐associated VITT (closed red circle), two spontaneous HIT (closed blue circles), three delayed‐onset HIT (closed yellow circles), eight classical HIT (open circles), and seven ELISA+/PEA‐ samples (open squares) to immobilized PF4‐polyanion complexes was evaluated using the LIFECODES PF4 IgG immunoassay (A) and the un‐complexed PF4 ELISA (B). Groups were compared using one‐way ANOVA. Mean and standard error are shown. ns, not significant (p = .5192); ***p < .001; ****p < .0001. Twenty‐five healthy control sera had a mean OD of 0.130 (range 0.075–0.233) in the un‐complexed PF4 ELISA (data not shown) [Color figure can be viewed at wileyonlinelibrary.com]