Abstract Number: OC 47.2
Meeting: ISTH 2021 Congress
Background: Technically-simple HIT ELISA assays are non-specific while complex functional assays have limited availability making timely, accurate diagnosis a challenge. A major barrier to widespread use of functional testing is the requirement for fresh platelets.
Aims: The goal of this study was to demonstrate proof-of-concept that functional testing can be developed as part of a technically-simple assay using long-term stored platelets.
Methods: Cryoprotectant was added to donor platelets prior to freezing, thawed after variable periods of time and incubated with platelet factor 4 (PF4) and serum from HIT-suspected patients. Thrombospondin-1 (TSP1, a highly expressed protein in platelet α-granules) released upon platelet activation was measured by ELISA. Studies were approved by the institutional ethics board.
Results: The TSP1-release assay (TRA) demonstrated that relative to healthy controls (open circles, Fig 1A), HIT sera (black circles) induced greater levels of platelet activation in all 20 frozen platelet preparations studied. Ten of the platelet preparations were tested again after 4 weeks and results obtained were similar (Fig 1B) confirming stability with storage. Sixteen samples from SRA-positive/ELISA-positive (black circles, Fig 2A), 8 SRA-negative/ELISA-positive (red squares) and 7 SRA-negative/ELISA-negative (black squares) patients were tested using frozen platelets. The TRA clearly separated SRA-positives from SRA-negatives with one exception (red circle), which was from a thrombotic “SRA-negative HIT” patient (PF4-Dependent P-selectin Expression Assay, PEA: 104%). To stringently assess TRA specificity, ELISA-matched groups of high optical density SRA-negative (open triangles; Fig 2B) and SRA-positive (closed triangles) samples were tested, and in contrast to ELISA, the TRA clearly separated SRA-positive from -negative patients.
Conclusions: Using a heterogeneous group of patient samples including those that were strongly false-positive in the HIT ELISA, the TRA demonstrated 100% sensitivity and specificity in pilot testing. These findings have the potential to transform the diagnostic testing paradigm in HIT by making near-patient functional testing available for rapid and accurate diagnosis.
To cite this abstract in AMA style:Padmanabhan A, Singh B, Jones C, Leger R, Pruthi R, Heikal N, Chen D. A Thrombospondin-1 Release Assay (TRA) Coupled to PF4-treated Frozen Platelets is Highly Accurate for the Detection of Pathogenic HIT Antibodies: Implications for Development of a Near-patient HIT in vitro Diagnostic Assay [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/a-thrombospondin-1-release-assay-tra-coupled-to-pf4-treated-frozen-platelets-is-highly-accurate-for-the-detection-of-pathogenic-hit-antibodies-implications-for-development-of-a-near-patient-hit-in/. Accessed November 27, 2021.
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