key: cord-0911366-mtk0mkoy
authors: Kanack, Adam J.; Singh, Bandana; George, Gemlyn; Gundabolu, Krishna; Koepsell, Scott A.; Abou‐Ismail, Mouhamed Yazan; Moser, Karen A.; Smock, Kristi J.; Green, David; Major, Ajay; Chan, Clarence W.; Wool, Geoffrey D.; Reding, Mark; Ashrani, Aneel A.; Bayas, Antonios; Grill, Diane E.; Padmanabhan, Anand
title: Persistence of Ad26.COV2.S‐associated vaccine‐induced immune thrombotic thrombocytopenia (VITT) and specific detection of VITT antibodies
date: 2022-02-21
journal: Am J Hematol
DOI: 10.1002/ajh.26488
sha: 7147473d2ddf0dd4ee8dd26d40ad38db519ed910
doc_id: 911366
cord_uid: mtk0mkoy

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 10(3)/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.

(140-150 x 10 3 /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. Studies on ChAdOx1 nCoV-19-associated VITT patients have demonstrated that the causative antibodies strongly recognize PF4-polyanion complexes [8] [9] [10] and require PF4-treated platelets (as opposed to heparin-treated platelets) for consistent detection by functional assays. 11 Much less is known about the characteristics of Ad26.COV2.S-associated VITT antibodies, including the duration of antibody persistence, information that would help define clinical and laboratory monitoring protocols in these patients. AD26.COV2.Sassociated VITT is rare, with an estimated incidence of 3.55 individuals per million vaccinated, 6 and is difficult to distinguish from another rare anti-PF4 antibody-mediated syndrome, spontaneous HIT. Spontaneous HIT, like VITT, is seen in the absence of proximate heparin exposure and has been referred to as the "background rate" of anti-PF4 antibody-mediated thrombotic thrombocytopenia in recent publications from the CDC. 6 Currently-available diagnostic assays fail to distinguish between VITT and spontaneous HIT (and "classical" HIT), as patients with these disorders have antibodies detectable by both HIT ELISAs (that have PF4-polyanion targets) and functional assays. 8, 12, 13 Furthermore, VITT and spontaneous HIT are clinically similar, characterized by severe thrombocytopenia and thrombosis, including in uncommon locations such as the cerebral venous sinus and splanchnic vasculature. 14, 15 In this study, we sought to assess VITT antibody persistence using currently available functional and antigen-based assays and to develop new laboratory tools for the specific detection of VITT that would allow its differentiation from classical and spontaneous HIT. 

A widely-used, FDA-approved in vitro diagnostic assay that uses PF4-polyvinyl sulfonate (PVS) targets, Lifecodes PF4 IgG (Immucor), was used according to manufacturer instructions (package insert on file). Briefly, similar to the un-complexed PF4 ELISA, patient serum/plasma incubation with PF4-PVS coated platelets was followed by stringent washing and incubation of an alkaline phosphatase labeled anti-human IgG antibody. pNPP substrate was then added, colorimetric detection was performed after 30 min of the reaction, and OD (405-492 nm) was recorded.

The PEA was performed as previously described. 16 Serotonin release assay (SRA), platelet count, and D-dimer testing were performed at various reference/hospital laboratories. Normal ranges for the Lifecodes PF4 IgG assay, SRA, and PEA were < 0.4 OD, <20% serotonin release, and <19% P-selectin expression, respectively. D-dimer reference ranges varied by laboratory (Table 1 ).

One-way ANOVA was used to compare the three groups of VITT, HIT, and ELISA+/PEA-patient samples. One patient who had a 

The Ad26.COV2.S-associated VITT patient cohort included five males and four females with a median age of 46 years (range 28-51; Table 1 ; blue) and platelets count (>150 x 10 3 /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]

All patients had strongly positive PF4-polyanion ELISA results (OD > 1.0), and eight patients demonstrated ODs > 2.0 ( Figure 1A ).

Two patients were tested in an automated, latex enhanced immunoturbidimetric assay (HemosIL HIT-Ab[PF4-H], Instrumentation Laboratory; data not shown), and both were falsely-negative. Eight of the nine VITT patients were tested by conventional serotonin release assay (SRA) using heparin-treated platelets, with four testing positive.

Available blood samples from six patients were tested in the PEA, a functional HIT assay that uses PF4 rather than heparin-treated platelets. 16 presentation. In contrast to ELISA, the probability of a positive PEA was very low 5 months after acute presentation ( Figure 1C ).

Platelets were mild-moderately decreased in three patients even after 5 months of follow-up (176, 194, and 152 days) at 141 x 10 3 /μL, 149 x 10 3 /μL, and 89 x 10 3 /μL, respectively. One of the three patients (with a platelet count of 89 x 10 3 /μL) had preexisting thrombocytopenia secondary to hepatic cirrhosis. Thus, it was deemed unlikely that the persistence of thrombocytopenia was due to VITT antibodies. Most patients had normalized their Ddimer levels within 30 days of presentation ( Figure 1D ).

To compare the current HIT ELISAs that employ PF4-polyanion targets and a novel un-complexed PF4 ELISA that uses PF4 in the absence of a polyanion for their abilities to distinguish between VITT, spontaneous HIT, and HIT, acute samples from six Ad26.COV2.S (5) and ChAdOx1 nCoV-19 (1)-associated VITT, two spontaneous HIT, eight "classical" HIT, and three delayed-onset HIT 17 patients were studied. Spontaneous HIT patients were heterogeneous in presentation, with one patient developing disease after knee arthroplasty and the other after several days of presumed infection characterized by nausea and fatigue. Both patients had severe outcomes due to thrombosis (above knee amputation and permanent neurological disability due to stroke, in the surgical and medical case, respectively). All three patients with delayed-onset HIT presented with heavy thrombotic burden and thrombocytopenia and needed readmission after being initially discharged after successful cardiac surgery. Table 2 presents platelet nadirs and the presence of thrombosis in these patients. The eight "classical" HIT patients had varied presentations with a platelet nadir range of 7-134 x 10 3 /μL and five of the eight patients developed thrombosis. Thrombosis was seen in all five patients with spontaneous HIT/delayed-onset HIT. As shown in Figure 2A , the PF4-polyanion assay was highly non-specific for the detection of VITT antibodies, with samples from all groups (VITT, spontaneous HIT, delayed-onset HIT, and HIT) producing strong reactions in this test.

An additional control group of seven ELISA+/PEA-samples demonstrated low but positive ODs in the PF4-polyanion ELISA, with some overlap with VITT samples. In contrast, when these cohorts were tested in an experimental ELISA assay using un-complexed rather than polyanion-complexed PF4 targets ( Figure 2B ), clear differentiation of VITT from all other groups was seen with anti-PF4 antibodies from VITT patients producing significantly higher optical densities relative to non-VITT groups. Of note, the sample from the ChAdOx1 nCoV-19-associated VITT patient 19, 20 included in these studies yielded high OD in this assay despite being drawn >2 months after initial presentation (4.00, Figure 2B ).

In this study, nine Ad26.COV2.S-associated VITT patients (15% of 57 cases reported to the CDC to date) were followed over a median The strong PF4-polyanion ELISA reactivity seen in our patients is consistent with recent findings in ChAdOx1 nCoV-19-associated VITT. 9 Like in ChAdOx1 nCoV-19-associated VITT, 8, 11 this study emphasizes the criticality of testing samples in PF4-enhanced functional assays, as heparin-based assays like the SRA can be falsely negative. These results also show that automated (non-ELISA) HIT assays should be avoided for Ad26.COV2.Sassociated VITT investigation due to poor sensitivity, as has been recommended for ChAdOx1 nCoV-19-associated VITT. 23 Ad26.COV2.Sassociated VITT antibodies were similar to ChAdOx1 nCoV-19-associated antibodies, 24 wherein antibodies positive in the PF4-polyanion ELISA persisted much longer relative to platelet-activating antibodies in PF4-enhanced functional assays. During recovery, the seroconversion profile of a negative platelet activation assay followed by a negative ELISA was similar to HIT 25 ; however, the duration of antibody positivity was significantly longer with VITT similarly, platelet count recovery occurs within 7 days in 90% of cases of HIT, 26 while it was significantly longer in our VITT cohort. In summary, patients with VITT demonstrate long-term ELISApositivity, although functional assays become seronegative earlier during the course of recovery. The data presented here suggest that the un-complexed PF4 ELISA is sensitive and significantly more specific for the detection of VITT than currently available tests; however,

given the condition's rarity and the small number of VITT and comparator HIT cohorts tested, future studies should further assess its diagnostic utility.

Thrombotic thrombocytopenia after Ad26.COV2.S vaccination

VITT following Ad26.COV2.S vaccination presenting without radiographically demonstrable thrombosis

Refractory vaccine-induced immune thrombotic thrombocytopenia (VITT) managed with delayed therapeutic plasma exchange (TPE)

Fatal vaccine-induced immune thrombotic thrombocytopenia (VITT) post Ad26.COV2.S: first documented case outside US. Infection

Successful treatment of thrombotic thrombocytopenia with cerebral sinus venous thrombosis following Ad26.COV2.S vaccination

Case Series of Thrombosis With Thrombocytopenia Syndrome After COVID-19 Vaccination-United States

Vaccine-induced thrombotic thrombocytopenia following Ad26.COV2.S vaccine in a man presenting as acute venous thromboembolism

Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination

Pathologic antibodies to platelet factor 4 after ChAdOx1 nCoV-19 vaccination

Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination

Adjunct immune globulin for vaccine-induced thrombotic thrombocytopenia

Use of intravenous immunoglobulin G to treat spontaneous heparin-induced thrombocytopenia

Spontaneous heparin-induced thrombocytopenia syndrome: 2 new cases and a proposal for defining this disorder

Spontaneous HIT syndrome: knee replacement, infection, and parallels with vaccine-induced immune thrombotic thrombocytopenia

Cerebral venous sinus thrombosis associated with spontaneous heparin-induced thrombocytopenia syndrome after total knee arthroplasty

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis

Delayed-onset heparin-induced thrombocytopenia and thrombosis

IVIg for treatment of severe refractory heparin-induced thrombocytopenia

Bilateral superior ophthalmic vein thrombosis, ischaemic stroke, and immune thrombocytopenia after ChAdOx1 nCoV-19 vaccination

Anti-PF4 VITT antibodies are oligoclonal and variably inhibited by heparin

Disease burden, complication rates, and health-care costs of heparin-induced thrombocytopenia in the USA: a population-based study

Clinical features of vaccine-induced immune thrombocytopenia and thrombosis

PF4 immunoassays in vaccine-induced thrombotic thrombocytopenia

Decline in pathogenic antibodies over time in VITT

Temporal aspects of heparin-induced thrombocytopenia

American Society of Hematology 2018 guidelines for management of venous thromboembolism: heparininduced thrombocytopenia

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia

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

We would like to thank Jill Kappers and Stephanie Hafner for their exceptional research coordination. This work was supported, in part, by National Institutes of Health grants HL158932 and HL133479 (Anand Padmanabhan).