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“Off-the-shelf” cryopreserved platelets for the detection of HIT and VITT antibodies

A. Kanack1, C. Jones2, N. Splinter3, R. Leger3, N. Heikal3, D. Chen3, R. Pruthi3, G. George4, Y. Abou-Ismail5, G. Wool6, K. Gundabolu7, A. Padmanabhan3

1Mayo cliinc, Rochester, Minnesota, United States, 2Retham Technologies, Wauwatosa, Wisconsin, United States, 3Mayo Clinic, Rochester, Minnesota, United States, 4Univ of Colorado, Denver, Colorado, United States, 5University of Utah, Salt Lake City, Utah, United States, 6University of Chicago, Chicago, Illinois, United States, 7University of Nebraska, Omaha, Nebraska, United States

Abstract Number: OC 02.3

Meeting: ISTH 2022 Congress

Theme: Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies » HIT

Background: Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are life-threatening disorders characterized by anti-Platelet Factor 4 (PF4) antibodies. Tests such as the PF4-polyanion enzyme-linked immunosorbent assay (ELISA) lack specificity, and accurate functional tests such as the serotonin release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) are technically complex and require fresh platelets; causing delays in diagnosis and overuse of alternative anticoagulants.

Aims: Develop a rapid, near-patient assay for diagnosing HIT and VITT using readily available “off-the-shelf” platelets.

Methods: Normal donor platelets were cryopreserved in a trehalose-based buffer. Thawed platelets were washed, treated with PF4 or heparin, and incubated with HIT or VITT samples before quantifying platelet thrombospondin-1 (TSP1) release in the TSP1-release assay (TRA). Results were expressed relative to TSP1 released by healthy donor serum-treated cryopreserved platelets.

Results: Platelet-activating (PEA-positive) samples stimulated a 2.52-fold mean increase in TSP1 release in the PF4-TRA (Fig 1A), and an average 2.79-fold increase in the heparin-TRA (Fig 1B), both significantly higher than TSP1 release induced by PEA-negative HIT-suspected patients (including some with false-positive ELISA results; red squares). Two non-activating samples induced TSP1 release in the heparin-TRA but were not inhibited by high-dose heparin (100 U/mL; data not shown), ruling out HIT. Cryopreserved platelets stored for >1 year supported detection of HIT antibodies (data not shown). Lastly, four VITT patient samples induced elevated TSP-1 release in the PF4-TRA (Fig 1A) but did not in the Heparin-TRA (Fig 1B).

Conclusion(s): The PF4- and Heparin-TRA were highly accurate for diagnosing HIT/VITT and HIT, respectively. VITT samples did not activate heparin-treated platelets, likely due to competition for the heparin-binding site on PF4. The use of “off-the-shelf” cryopreserved platelets has the potential to transform the diagnostic testing paradigm by making near-patient functional testing available for the rapid and accurate diagnosis of HIT and VITT.

To cite this abstract in AMA style:

Kanack A, Jones C, Splinter N, Leger R, Heikal N, Chen D, Pruthi R, George G, Abou-Ismail Y, Wool G, Gundabolu K, Padmanabhan A. “Off-the-shelf” cryopreserved platelets for the detection of HIT and VITT antibodies [abstract]. https://abstracts.isth.org/abstract/off-the-shelf-cryopreserved-platelets-for-the-detection-of-hit-and-vitt-antibodies/. Accessed August 9, 2022.

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