PB0993 - Measurement of procoagulant platelets in platelet-rich plasma by flow cytometer for the diagnosis of heparin-induced thrombocytopenia

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

LP

Lisann Pelzl

Post Doc
Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty of Tuebingen, University Hospital of Tuebingen, Tuebingen, Germany
Tuebingen, Baden-Wurttemberg, Germany

Co-Author(s)

AK

Aleyna Karakuyu

student
Experimentelle und Klinische Transfusionsmedizin Tübingen, Germany
Tuebingen, Baden-Wurttemberg, Germany
Jan Zlamal

Resident/PhD Student
Institute for clinical and experimental transfusion medicine Tübingen
Tübingen, Baden-Wurttemberg, Germany
IM

Irene Marini

PhD
Center for Clinical Transfusion Medicine
Tübingen, Baden-Wurttemberg, Germany
AS

Anurag Singh

Postdoc
Experimentelle und Klinische Transfusionsmedizin Tübingen
Tuebingen, Baden-Wurttemberg, Germany
HJ

Hisham Jaffal

Student
Institute for clinical and experimental transfusion medicine Tübingen
Tuebingen, Baden-Wurttemberg, Germany
Stefanie Hammer, MD

Physician
Center for Clinical Transfusion Medicine
Tübingen, Baden-Wurttemberg, Germany
Karina Althaus, MD

Physician, Director of Platelet Immunology Lab.
Center for Clinical Transfusion Medicine, University Hospital of Tübingen
Tübingen, Baden-Wurttemberg, Germany
Tamam Bakchoul, MD

Medical Director
Center for Clinical Transfusion Medicine, University Hospital of Tübingen
Tübingen, Baden-Wurttemberg, Germany

Background: Heparin-induced thrombocytopenia (HIT) is caused by anti- PF4/heparinIgG antibodies, which activates platelets and leads to thrombocytopenia and thrombosis. The diagnosis of HIT can only be confirmed using functional assays such as the Heparin-Induced Platelet Activation assay (HIPA assay). However, functional assays are technically demanding and routinely available only in specialized laboratories.

Aims: The aim of the current study was to establish a flow cytometer-based method to detect procoagulant platelets in platelet-rich plasma (PRP) for the diagnosis of HIT.

Methods: Sera samples from patients with HIT (HIT group) were incubated with PRP from healthy donors for different durations with (30, 60, 90 minutes). Procoagulant platelets were determined by double expression of P-selectin (CD62p) and phosphatidylserine (PS) externalization by flow cytometry. CD32a-mediated cross-link and platelet stimulation with ionomycin were used as positive controls.

Results: Sera from HIT-diagnosed patients but not from the control-group induced a significant increase in the procoagulant platelet subpopulation in the presence of 0.2 U/mL heparin (% double positive CD62/PS: 1.2±1.1 vs 18.5±8.1, p=0.0021). The optimal incubation time was 60 minutes. A donor dependency of the flow cytometric method was not observed (control vs HIPA+: 0.7±0.59 vs19.0±3.2, p=0.0129). In addition, the use of washed platelets and PRP with HIT-sera led to the same results in the flow cytometric method (34.2±6.3 vs 30.1±2.2, ns).

Conclusion(s): Our data suggest that PRP-based protocol can be used to detect the ability of HIT antibodies to induce procoagulant platelets by flow cytometry. Ongoing study is currently investigating the clinical implementation of this protocol in the diagnostic work up for HIT.