Leger, Rachel

Abstract Objectives To determine the impact of residual platelets on dilute Russell’s viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. Methods We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. Results Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. Conclusions Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

AbstractHeparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital 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 Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14–labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

Abstract Introduction: Heparin-induced Thrombocytopenia (HIT) is a potentially life and limb threatening pro-thrombotic syndrome triggered by antibodies to platelet factor 4 (PF4)/polyanion complexes. Commonly used screening tests such as the PF4/Heparin ELISA (PF4 ELISA), while easy to perform in the near-patient setting, are highly non-specific. In contrast, more accurate tests such as the serotonin release assay (SRA) are technically complex, require radioactivity and are performed in only a handful of reference laboratories. These diagnostic challenges lead to both unnecessary anticoagulant use as well as avoidable cases of severe bleeding. Recent reports suggest that a relatively simple three-hour flow cytometry-based test that uses PF4-treated normal platelets, the PEA (PF4-dependent p-selectin Expression Assay), is at least as accurate as the SRA for identifying patients who have HIT (Thromb Haemost. 2015 Nov 25;114(6):1322-3; Chest. 2016 Sep;150(3):506-15; Chest. 2017 Oct;152(4):e77-e80). Here we describe results of a large prospective study designed to compare the accuracy of the SRA and PEA for HIT diagnosis in a clinical setting. Methods: The PEA and SRA were performed on serum samples obtained from adult patients (≥18 yrs.) from 2 large tertiary care academic hospitals from May 2016 to Sep 2017. All inpatients for whom PF4 ELISA testing was ordered were included in the study. The PEA was performed with platelets pretreated with PF4 at 3 concentrations: 30mcg/mL (PEA30), 10mcg/mL (PEA10) and 3mcg/mL (PEA3). Sera were batch-tested in both assays (SRA and PEA) using the same target platelets. The central laboratory was blinded to clinical history and to the results of the PF4 ELISA performed by participating hospitals at the time of diagnosis. Both centers performed IgG-specific PF4 ELISAs and utilized manufacturer-recommended cut-offs for positivity. Predefined "more-stringent" and "less stringent" criteria based on clinical findings (4Ts score) and results of PF4 ELISA testing were used to define the likelihood of a patient having HIT (Table 1A). Receiver Operating Curve (ROC) analysis was performed and Area Under the Curve (AUC) of each assay was calculated to assess their diagnostic accuracy. Since high test specificity was desired, positive cut-offs that maximized the sum of sensitivity and specificity in a specificity range of ≥90% and ≥95% were derived. To evaluate non-inferiority of the PEA to the SRA, we assessed whether the lower limit of the one-sided 95% confidence interval of the ΔAUC (PEA-SRA) was higher than a non-inferiority margin of 5% and 10%. Results: A total of 440 patient samples were tested with both assays. Seventeen and 28 patients were classified as having true HIT based on the use of the more- and less-stringent criteria, respectively. When classified by more-stringent criteria, the AUC was numerically greater for each of the three PEA tests than for the SRA (Table 1B). All tests had excellent accuracy (AUC>0.90, "excellent" by the Traditional academic point system). Using less-stringent criteria, the PEA30 had the highest AUC estimate followed by the SRA (Table 1C). Positive cut-offs generated at test specificities of ≥90% and ≥95% using data classified by more stringent criteria are shown in Table 1D. PEA3 was non-inferior to the SRA at a 5% non-inferiority margin, and all PEA tests were non-inferior at the 10% margin using the more stringent classification for true HIT (Fig 1A). In the less-stringently classified data set, the PEA30 was non-inferior to the SRA at a 10% margin (Fig 1B). Conclusions: The PEA, a 3-hour, non-radioactive functional HIT assay demonstrated high diagnostic accuracy and was non-inferior to the SRA using commonly accepted non-inferiority margins for diagnostic testing (5-10%). The technical advantages of the PEA over the SRA may encourage its widespread adoption as a rapid-turnaround, accurate and near-patient diagnostic for patients with suspected HIT. Disclosures Jones: BloodCenter of Wisconsin: Patents & Royalties. Curtis:Ionis Pharmaceuticals: Consultancy. Bougie:BloodCenter of Wisconsin: Patents & Royalties. Aster:BloodCenter of Wisconsin: Patents & Royalties. Garcia:Shingoi: Consultancy; Retham Technologies LLC: Consultancy; Incyte: Research Funding; Daiichi Sankyo: Research Funding; Bristol Meyers Squibb: Consultancy; Portola: Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy; Boehringer Ingelheim: Consultancy. Padmanabhan:BloodCenter of Wisconsin: Patents & Royalties: Patent/royalties for HIT diagnostic testing; Veralox Therapeutics: Other: Advisory Board; Retham Technologies LLC: Equity Ownership, Other: Retham seeks to develop HITDx(TM), a new assay for HIT diagnosis; TerumoBCT: Consultancy; GE Healthcare: Other: Material support for Clinical Quality Improvement Initiative; Janssen Pharmaceuticals: Consultancy.