Researchers have developed a flow cytometry-based method to assist with diagnosis of vaccine-induced immune thrombotic thrombocytopenia (VITT). They recently described the assay and its use in a study with results reported in the journal Blood Advances.

“Early and accurate diagnosis of potentially fatal VITT has far-reaching implications for public health and clinical management, as early intervention significantly improves patient outcomes,” the study investigators wrote in their report.

VITT is thought to involve thrombosis and thrombocytopenia through an FcgRIIa-dependent pathway, the study investigators explained in their report. The detected of anti-platelet factor 4 (PF4) antibodies associated with the condition is sometimes used to diagnose VITT. “Not all immunologically detected anti-PF4 antibodies are pathologic, with 6.8% of anti-PF4 antibodies detectable by screening of well individuals after COVID-19 vaccination,” the study investigators wrote in their report. They set out to develop a functional assay for identifying VITT.

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The assay described in the study utilizes flow-cytometric detection of procoagulant platelets (PPs), which develop as a response to antibodies in VITT. The assay identifies PPs by measurement of P-selectin and a cell death marker called 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, or GSAO.

Plasma was evaluated with this assay using whole-blood samples from Australian patients who were referred for confirmation of VITT. VITT was diagnosed through exposing whole blood from healthy volunteers to patient plasma that had been treated with a platelet agonist. In vitro experiments were performed to evaluate procoagulant responses.

Samples from 47 patients in whom VITT was suspected were used for assay development. A significant increase in the level of PPs was seen in plasma from 23 patients who had suspected VITT based on immunoassays. However, PPs were not significantly increased in plasma from 32 volunteers without the disease or from 24 individuals who had received the ChAdOx1 nCov-19 vaccine but without developing evidence of VITT.

A procoagulant response was reportedly induced in donor platelets exposed to plasma from patients with suspected VITT, with this response suppressed by heparin. PP response appeared mediated through FcgRIIa, based on inhibition by intravenous immunoglobulin (IVIg). In vitro experiments using IVIg and fondaparinux showed effects on PP levels that trended with clinical responses to these agents.

A validation cohort consisted of 99 patients with adjudication-confirmed VITT, and 98 of these patients were evaluated using the assay developed in this study. Of these patients, VITT was identified in 93%. The formation of PP appeared positively correlated with levels of anti-PF4 antibodies (r, 0.406; P <.0001), as well as D-dimer levels (r, 0.315; P =.002), and it was negatively correlated with platelet count (r, -0.386; P <.0001).

“In conclusion, induction of FcgRIIa-dependent PP responses by patient plasma that were suppressible by heparin and IVIg is highly indicative of VITT in the correct clinical circumstance,” the study investigators wrote in their report.


Lee CSM, Liang HPH, Connor DE, et al. A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia. Blood Adv. 2022;6(11):3494-3506. doi:10.1182/bloodadvances.2021006698