Citations

Background: Conduction tissue in the ventricles is closely intertwined with myocardium. Owing to high velocity of conduction, there is near simultaneous activation , making it difficult to discern. Restricted sampling rates and limited dynamic range make real time signal processing challenging for these signals in routine recording systems. Objectives: We aimed to use novel signal processing techniques in a new system to isolate the conduction tissue signals from the rest of the myocardial activation. Methods: We used the PURE-EP™ (BioSig Technologies, MN) signal processing system to record endocardial and epicardial signals from 6 anesthetized canines in an acute study setting. A novel filter based on proprietary algorithms to isolate the high frequency signal was applied at sites where the electrogram signals were thought to have a conduction tissue component to them. . In addition, ventricular fibrillation (VF) was induced at the end of the experiment and signals were then recorded from the endocardium and the conduction system. Results: This filter was successfully able to isolate conduction tissue signals in the myocardium from those of the ventricular muscle. It did over sense atrial signals at the basal left ventricular septum owing to the high frequency components of the atrial signal at this location. When applied to epicardial signals, it did not pick up any conduction tissue signals. In VF, we could discern signals originating from the conduction system endocardially. (Figure 1) Conclusions: This filter can isolate conduction tissue signals from those of the myocardium and may prove to be a useful adjunct in mapping arrhythmias originating near the conduction system.

Background: Transcatheter aortic valve replacement (TAVR) is often complicated by AV block requiring pacing, but whether patients recover AV conduction and whether the need for pacing affects outcomes is unknown. Methods: We retrospectively studied 534 patients who underwent TAVR at Mayo Clinic in Rochester, Minnesota between November 2008 and January 2015. Of these, 115 patients had implantable cardiac devices. Data was collected from subsequent device follow-ups to determine pacemaker dependency and percentage pacing. Results: 44 patients (8.2%) had devices implanted in the immediate post procedural period. The most common indications for implantation were high grade AV block (18 patients) and complete heart block (14 patients). 17/44 (38.6%) patients were pacemaker-dependent at implant and only 4 of these patients were no longer pacemaker-dependent during subsequent followup. The indication for pacing among these four patients was high grade atrio-ventricular block (2 patients), asystole (1 patient) and complete heart block (1). Among non-pacemaker-dependent patients, no significant change in the ventricular pacing percentages was noted over time. Survival was similar between patients who required device implantation post TAVR and those who did not after adjustment for age and gender (HR 0.73 [0.39-1.36], p=ns; figure) Conclusion: Patients requiring pacing after TAVR are often pacemaker-dependent at the time of implant. It is uncommon for patients who are dependent at the time of implant to regain AV conduction, but the need for cardiac pacing is not associated with increased mortality.

AbstractRare 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.

Abstract Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis. Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry. Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation. Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D). Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis. Figure Legend: Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. Figure 1 Figure 1. Disclosures Pruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Heparin induced thrombocytopenia (HIT) is a rare immune mediated complication that is triggered in a subset of patients temporal to therapeutic heparin exposure. Laboratory testing is based on screening for the presence of serum anti-PF4 antibodies using sensitive solid-phase immunoassays. If antibodies are detected, functional testing to demonstrate the platelet activating properties and heparin dependence of these immune complexes is then performed. Previous studies have reported possible clinical utility in identifying non heparin dependent platelet activating antibodies using a buffer control with zero heparin in the serotonin release functional assay (SRA). These reports suggested a correlation between reactivity in the zero heparin buffer control and pathogenicity of HIT antibodies which may define a subtype of HIT, referred to as autoimmune HIT. We aimed to investigate the utility of zero heparin buffer control as a part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA). Methods: Three hundred archived serum samples were tested using anti-PF4 IgG antibody enzyme-linked immunosorbent assay (ELISA; Immucor Diagnostics, GA, USA). SRA was preformed on all samples using Mayo-SRA and reference 14C SRA methods. Zero heparin control buffer was included in the Mayo-SRA assay. Serotonin release >20% in the low dose heparin (0.1U/mL, LDH) and ELISA optical density (OD) >0.4 were considered positive. Drug interference studies were performed by spiking known SRA-positive samples with increasing concentrations of unfractionated heparin (UFH), low molecular weight heparin (LMWH) or fondaparinux in the LDH and zero heparin SRA. The clinical 4T score was calculated retrospectively calculated for all patients. Results: Of the 300 tested samples, 57 were anti-PF4 ELISA positive. 33 of the 57 samples were positive using the reference 14C SRA method. Whereas 43 samples were positive by Mayo-SRA assay. Three additional samples were positive by Mayo-SRA, but negative by both screening anti-PF4 ELISA and the reference SRA method (Fig- 1A). Lastly, 13 samples were anti-PF4 ELISA positive, but SRA negative by both methods in comparison. Interestingly, 44 of 46 (95%) samples interpreted positive by LDH were also interpreted positive (serotonin release >20%) under zero heparin conditions. These included the 3 samples that were positive by Mayo-SRA but negative by both screening anti-PF4 ELISA and the reference SRA. The overall % serotonin release using zero heparin control was significantly lower (P= 0.003, paired Student T-test) compared to LDH (Fig-1B). In addition, zero heparin followed a similar pattern as LDH, with highest levels at ELISA OD units >2 (Fig-1C). Strikingly, drug interference studies showed artifactual serotonin release in the zero heparin reaction, which was not detected in the absence of spiked drugs. For UFH, serotonin release in zero heparin control occurred at very low spiked concentrations, ≥0.063 U/mL. LMWH and fondaparinux spiking experiments also displayed similar zero heparin serotonin release patterns (Fig-1D). Of note, none of these interferences were detected in UFH spiked SRA-negative samples (data not shown). Conclusion: Contrary to prior results suggesting that less than 50% of LDH SRA positive samples are also positive in the zero heparin SRA, our results show high zero heparin SRA positivity rate of >95%. Zero heparin SRA showed a pattern with highest levels at ELISA OD units >2 suggesting that reactivity in this condition is a function of antibody strength rather than a qualitative difference (i.e. "autoimmune" HIT antibodies vs "non-autoimmune" HIT antibodies). In addition, contamination of patient sera with small amounts of remnant heparin can significantly impact platelet activation in the zero heparin SRA test. Thus, zero heparin SRA positive results may be artifactual and represent residual heparin contained in the patient sample. Figure legend: Fig-1A. Scatter plots of SRA positive samples grouped by 4T scores. Red dots are Mayo-SRA only positive samples. Fig-1B and Fig-1C. Scatter plots of % serotonin release of Mayo-SRA positive samples at LDH (circles) vs. zero heparin buffers (squares), and grouped by ELISA OD values, respectively. Fig-1D. % Serotonin release of known SRA-positive samples spiked with increasing concentrations of UFH, LMWH, or fondaparinux at LDH (white) or zero heparin buffers (red). Figure 1 Figure 1. Disclosures Pruthi: CSL Behring: Honoraria; Genentech: Honoraria; Bayer Healthcare AG: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening condition that could occur following exposure to heparin. Accurate and timely diagnosis is critical for appropriate clinical management. Laboratory testing for suspected cases is based on screening for the presence of serum anti-PF4/heparin antibodies using solid-phase enzyme-linked immunosorbent assay (ELSIA) which is known to be sensitive but less specific. A positive ELISA test is followed by functional testing to demonstrate the platelet activating properties and heparin dependence of the pathogenic antibodies. Serotonin release assay (SRA) is considered the gold standard functional test for the diagnosis of HIT. In most anti-PF4/heparin ELISA assays, a high-dose heparin buffer (100U/mL) confirmation step is recommended to demonstrate heparin dependence of detected antibodies and increase the specificity of the assay. The necessity of this confirmation step is controversial with some reports suggesting it could lead to misinterpreting positive ELISA results as negative or indeterminate, especially in cases of very strong and high titer HIT activating antibodies. We hence aimed to investigate the utility of applying this confirmation step as part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA). Materials: Three hundred archived serum samples were tested using anti-PF4/heparin IgG antibody ELISA (Immucor Diagnostics, GA, USA). High-dose heparin (100U/mL) confirmation step was performed on all samples with OD units ≥0.4 as recommended by the manufacturer. Samples with OD ≥ 0.4 and ≥50% OD inhibition in the high dose heparin confirmation step are interpreted positive. Mayo-SRA results were compared to a reference 14C SRA method. The 4T clinical score was retrospectively calculated for all patient (range 0-8 points). Results: Of the 300 tested samples, 57 samples were interpreted positive by the anti-PF4/heparin screening ELISA. 33 of the 57 samples were positive using the reference 14C SRA method, whereas 43 samples were positive by Mayo-SRA assay (≥20% serotonin release). Three additional samples were positive by Mayo-SRA, but negative by both screening ELISA and the reference 14C SRA method. All samples with OD units ≥0.4 displayed >50% inhibition in the high-dose heparin regardless of the intensity of the initial OD value or the HIT 4T score, with the exception of one that was negative by both SRA methods and of 1.35 OD value and 6 4T HIT score (Fig-1A). Importantly, thirteen samples were anti-PF4/heparin positive, but SRA negative (by Mayo-SRA and reference method). These samples also displayed positive %heparin inhibition (≥50% OD inhibition) (Fig-1B). Lastly, there were no differences in the degree of %inhibition in samples positive by both reference and Mayo-SRA or Mayo-SRA only (Fig-1B). Conclusion: In our patient cohort, addition of the high dose heparin inhibition confirmation step to the screening anti-PF4/heparin ELISA assay was of no additional diagnostic utility. We hence propose eliminating the heparin inhibition step which would improve laboratory turnaround time, reduce costs, and importantly speed up urgent clinical management decisions. Figure Legend: Fig-1A. No correlation between initial OD values and %OD inhibition using high dose heparin. Scatter plot of all ELISA positive samples (OD ≥0.4) grouped according to OD values. Samples include all SRA-positive and thirteen SRA-negative ELISA-positive samples. Fig-1B. Scatter plot of %OD inhibition comparing samples positive by Mayo-SRA and reference SRA method, positive by Mayo-SRA only, and ELISA-positive SRA-negative by both methods. Figure 1 Figure 1. Disclosures Pruthi: Bayer Healthcare AG: Honoraria; CSL Behring: Honoraria; Merck: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract VITT is an immune-based complication of adenoviral-based vaccines used to immunize against SARS_CoV2. The antibodies in VITT have been described as directed at the platelet-specific chemokine PF4 (CXCL4). While the clinical course and target chemokine in VITT has much in common with the better-known thrombocytopenic/prothrombotic disorder, heparin-induced thrombocytopenia (HIT), which involves antibodies directed against PF4 bound to the polyanion heparin, the specific loci where VITT and PF4/polyanion HIT antibodies bind appear to differ in studies using alanine-scanning mutations of PF4 (Nature, 2021. DOI: 10.1038/s41586-021-03744-4). The VITT antigenic site localizes to a heparin-binding domain. Unlike the dominant HIT locus, the VITT locus is conserved not only between human and mouse PF4, but also between PF4 and the related platelet-specific chemokine NAP2 (CXCL7). NAP2 is also expressed and stored in platelet alpha-granules and is present in equimolar concentrations to PF4. Unlike PF4, NAP2 avidly binds the chemokine receptor CXCR2 and strongly activates neutrophils. We now show that antibodies from patients who developed VITT after both AstraZeneca (AZ) or Johnson and Johnson (JJ) adenoviral vaccines, unlike HIT antibodies, recognize mouse PF4 (Figure 1A). More importantly, both AZ and JJ VITT antibodies bound NAP2, while none of the HIT antibodies tested bound PF4 or NAP2 in the absence of heparin (Figure 1A). These results are consistent with the alanine-scanning studies that distinguish the HIT and VITT binding sites. Dynamic light scattering (DLS) showed that NAP2 and PF4 bind to the adenoviral vectors, including Ad5 and the AZ vector ChAdOx5, which leads to expression of SARS_CoV2 spike protein. ChAdOx2 vaccine and CsCl 2-purified ChAdOx2 bound to both proteins, but form larger complexes with NAP2 than with PF4 even at lower concentrations of this chemokine (Figure 1C). Removal of anti-PF4 antibodies by hPF4-Sepharose abrogated PF4-dependent binding, but did not significantly reduce binding to NAP2 (not shown), indicating that VITT plasma contains discrete pools of anti-PF4 and anti-NAP2 antibodies that may have distinct functional properties. Sandwich ELISA (not shown) and Western blot analysis of purified VITT IgG demonstrates the presence of hPF4-IgG and NAP2-IgG immune complexes in purified patient's IgG (Figure 2A). Functional studies show that both PF4 and NAP2 can activate platelets in the presence of VITT antibodies. Anti-PF4-depleted VITT IgG fraction retains the ability to activate platelets in the presence of NAP2 (Figure 2B). Thus, unlike HIT, VITT appears to target a shared antigenic site on the related chemokines PF4 and NAP2. This raises the question as to whether NAP2, as one the most abundant platelet chemokines released from activated platelets, is involved in the initiation and propagation of the immunothrombotic response. Additional studies are needed to see whether NAP2, which can potently and specifically activate neutrophils via CXCLR2, contributes to the specific thromboinflammatory phenotype seen in VITT. We propose using FcgammaRIIA+ mice that concurrently express human PF4 and NAP2 and specific knockout of each chemokine, available in our group, to further understand the pathogenesis of VITT and its thrombocytopenic/ prothrombotic phenotype. Figure 1 Figure 1. Disclosures Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees. Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.