ITT therapy. 4,5 Treatment of SRA-confirmed HIT with fondaparinux resulted in mixed outcomes, 23 and similar "resistance" may also occur in VITT. We compared the effect of exogenous fondaparinux on PP response induced by   pretreatment plasma from 6 fondaparinux-treated patients with VITT. Inhibition of the PP response was reported at control concentrations aimed to disrupt charge-related interactions (100 mg/mL fondaparinux). At the therapeutic fondaparinux concentration (1.2 mg/mL), we observed a decreasing trend in PP response induced by plasma from 4 patients who clinically responded to fondaparinux therapy ( Figure 2F). In contrast, no change in PP response was seen with fondaparinux when tested with plasma from patients who did not respond clinically. No relationship between response and ELISA optical density was detected (supplemental Figure 1).

Addition of ChAdOx1 nCoV-19 and SARS-CoV-2 spike protein resulted in an inconsistent effect on procoagulant response
Exogenous ChAdOx1 nCoV-19 did not induce a procoagulant response in the absence of VITT plasma (supplemental Figure 2). Addition of ChAdOx1 nCoV-19 enhanced PP response in some, but not all, patients with VITT ( Figure 2G).

Diagnostic utility of PP assay
To evaluate the diagnostic potential, a receiver-operating characteristic curve was generated using plasma from patients with confirmed VITT (n 5 23) who tested positive on both ELISA and SRA, and VITT-negative patients (n 5 24) comprising ELISA-negative and SRA-negative patients, and ELISA false-positive patients who were non-thrombocytopenic and SRA negative. The area under the curve was 0.97 6 0.02 (P,.0001) with a cutoff of 1.7-fold increase in plasma-induced PP formation demonstrating 100% sensitivity and 91.7% specificity ( Figure 3A; supplemental Table 1). Suppression of PP response by IV.3-mediated blockade of FcgRIIa may further discriminate between antibody-mediated (VITT) and non-antibodymediated (non-VITT) procoagulant response. A,0.80-fold change with IV.3 achieved 100% sensitivity and 91.3% specificity, with an area under the curve of 0.99 6 0.01 (P,.0001) ( Figure 3B; supplemental Table 2) and identified individuals with non-antibodymediated PP enhancement. Figure 3C-H presents the patterns of PP response used in reporting. A pattern we termed "classical VITT" shows the increase in PP compared with baseline with reduction in the presence of 0.5 U/mL heparin, abrogation in the presence of 100 U/mL heparin, and at least a 0.80-fold decrease with IV.3. The pattern we termed "heparin-enhancing VITT" exhibits the same as classical VITT but with an increased PP response in the presence of 0.5 U/mL

Pattern of PP response in VITT
heparin. An equivocal (non-antibody-mediated procoagulant) response shows an increase in PP but without reduction with IV.3 regardless of other reactivity.

Evaluation of utility of standard flow cytometry assay in case ascertainment
Analysis of 108 cases referred for functional testing within the first 2.5 months of VITT in Australia (Table 2) found 59 cases consistent with VITT. No "VITT-negative" patients had a positive flow cytometry assay. Of the 59 patients with VITT, results of 41 cases were concordant between ELISA, standard SRA, and flow cytometry. Four patients who tested positive on both standard SRA and flow cytometry were ELISA negative but classified as VITT by the THANZ VITT advisory group who would have been missed using ELISA criteria alone. Fourteen patients were positive by standard flow cytometry who were missed by initial standard SRA testing. Thirteen of 14 were VITT serologically supported by either ELISA, PF4-enhanced SRA, or MEA as functional assays indicating likely true positive findings. One of 14 was clinically adjudicated to be VITT without additional serologic support. This standard flow cytometry assay enabled 7.4% cases in this cohort to be classified as VITT that would otherwise be missed by ELISA and 14.8% cases missed by standard SRA.
Addition of PF4 in clinically high probability cases that are negative in standard flow cytometry Addition of exogenous PF4 has shown improved sensitivity of functional platforms to detect platelet-activating antibodies in VITT. 1,12,25,26 We investigated the effect of purified human PF4 on performance of our flow cytometry assay using plasma from confirmed VITT-positive and VITT-negative cases. Exogenous PF4 did not alter results of VITT-positive (n 5 6) or VITT-negative (n 5 8) cases reported by standard flow cytometry assay without PF4 ( Figure 4A). Plasma samples that were inconclusive or negative in our standard assay, but positive via SRA or PF4-SRA, were tested with exogenous PF4 addition to flow cytometry. Two of 3 patients who were equivocal or inconclusive according to standard flow cytometry became positive, whereas one remained equivocal after PF4 addition ( Table 2). Three of 5 patients who were thought to be false negative on standard flow cytometry (SRA positive) were labeled positive with PF4-enhanced flow cytometry.

Performance of flow cytometry assay in cases referred for VITT testing
Of 99 cases adjudicated as VITT by the THANZ VITT advisory group from 1 April 2021 to 7 September 2021 (Table 3), 98 underwent flow cytometry testing. Four patients had no serologic support by either ELISA or any functional test platform. Seventy-six of the remaining 94 were positive on flow cytometry without PF4 enhancement. Fifteen of 18 individuals classified as negative, inconclusive, or equivocal returned a positive result with PF4 enhancement ( Figure 4B). Three remained equivocal (increased procoagulant response to plasma without IV.3 suppression). Overall, 93% of patients with clinicopathologic adjudication of VITT were detectable by flow cytometry assay with sequential testing of negative and equivocal samples with addition of exogenous PF4. PP formation showed a positive correlation with anti-PF4 antibody (r 5 0.406; P,.0001) ( Figure 4C) and D-dimer (r 5 0.315; P 5.002) ( Figure 4D) levels, and negative correlation against platelet count (r 5 -0.386; P,.0001) ( Figure 4E).

Discussion
Mass vaccination is imperative to battle COVID-19, but rare thrombotic events associated with SARS-CoV-2 vaccination raise concerns regarding adenoviral vector vaccine safety. 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. We previously established a whole-blood flow cytometry-based PP assay for HIT diagnosis that exploited the synergistic procoagulant effect of activating platelet GPCR and ITAM signaling pathways. 20 Considering the pathophysiological similarities between HIT and VITT, we adapted our assay to ascertain diagnostic utility in a development cohort of 47 patients suspected of VITT and confirmed this within 290 sequential patients referred for VITT functional testing. This assay has been incorporated into the diagnostic strategy of VITT in Australia.
As hypothesized, the level of PP formation with low-dose SFLLRN stimulation was minimal in healthy donor whole blood, despite near complete a-granule release. There was no increase in FcgRIIadependent procoagulant response with the addition of negative control plasmas that lack ITAM receptor engagement, whereas plasma with HIT or VITT antibodies capable of activating ITAM signaling exhibited a procoagulant response consistent with the known synergistic effect of combined GPCR and ITAM signaling on formation of this platelet subpopulation.
Evaluation using plasma samples from ELISA-positive, SRA-positive VITT patients confirmed that our assay detected plasma-induced FcgRIIa-dependent PP responses in donor platelets suppressible by high-dose heparin and IVIg. The procoagulant response was not observed in vaccinated patients without anti-PF4 antibodies and SRAnegative, or those with detectable nonpathologic anti-PF4 antibodies (ELISA-positive, SRA-negative, non-thrombocytopenic patients), demonstrating specificity. Inclusion of criterion for suppression of plasmainduced procoagulant activity by IV.3 increased assay specificity. The heightened PP response generated by VITT plasma was positively associated with anti-PF4 antibody levels and trended with decreasing platelet count at time of diagnosis. Extent of thrombocytopenia has been associated with significant mortality risk whereby for every 50% reduction in baseline platelet count there is a 1.7-fold increased risk of VITT-related death. 27 Two other flow cytometry assays have been described that detect VITT antibodies: PF4-induced flow cytometry-based platelet activation by Handtke et al, 28 performed on hirudinated donor whole blood with addition of PF4 with P-selectin MFI as output; and a washed platelet PP assay, by Althaus et al, 12 again supplemented with PF4, with combined annexin V binding and P-selectin expression as readout. Our assay of PP response induced by patient plasma in citrated whole blood from responsive donors represents an additional functional platform to heparin-induced platelet activation, SRA, PF4-induced flow cytometry-based platelet activation, and MEA.
Increasingly, data suggest that VITT is a spectrum disorder. There is variation in anti-PF4 antibody levels, and one quality assessment exercise revealed significant variability in functional assay VITT antibody detection, showing the need for ongoing refinement of VITT diagnostic assays.