OC 02.3 - “Off-the-shelf” cryopreserved platelets for the detection of HIT and VITT antibodies

Saturday, July 9, 2022

1:30 PM – 1:45 PM

Location: S11B

Presentation Format: In-Person

Presenting Author(s)

adam J. Kanack, PhD

Post doctoral fellow
Mayo Clinic
rochester, Minnesota, United States

Co-Author(s)

CJ

Curtis Jones

Director, Research & Development
Retham Technologies
Wauwatosa, Wisconsin, United States
NS

Noah Splinter

Research Technologist
Mayo Clinic
Rochester, Minnesota, United States
RL

Rachel Leger

Senior Developer
Mayo Clinic
Rochester, Minnesota, United States
NH

Nahla Heikal

MD
Mayo Clinic
Rochester, Minnesota, United States
DC

Dong Chen

MD PhD
Mayo Clinic
Rochester, Minnesota, United States
RP

Rajiv Pruthi

MBBS
Mayo Clinic
Rochester, Minnesota, United States
GG

Gemlyn George

Physician
Univ of Colorado
Denver, Colorado, United States
YA

Yazan Abou-Ismail

Physician
University of Utah
Salt Lake City, Utah, United States
GW

Geoffrey Wool

MD PhD
University of Chicago
Chicago, Illinois, United States
KG

Krishna Gundabolu

Physician
University of Nebraska
Omaha, Nebraska, United States
anand padmanabhan, MD

Associate Professor
Mayo Clinic
Rochester, Minnesota, United States

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.