Platelet Microparticle Generation Assay: a Valuable and Simpler Alternative to 14C-SRA for Type-II HIT Diagnosis.
Authors: Mullier François , Bailly Nicolas , Cornet Yvan , Robert Severine , Osselaer Jean-Claude , Chatelain Christian , Elalamy Ismail , Dogné Jean-Michel , Chatelain Bernard 
Published: October 2019

Abstract
Abstract 1442

Introduction:
Type II heparin-induced thrombocytopenia (HIT) is a serious immune-mediated adverse effect of heparin treatment. The release of procoagulant platelet microparticles (PMPs) is considered as major component of this thrombogenic disease. Early diagnosis of HIT is essential in order to improve clinical outcomes. However, such a diagnosis is challenging. The current diagnostic tests consist of the combination of the clinical scoring system (“4T's rule”) with serological or functional tests such as the gold standard 14C-serotonin release assay (14C-SRA). Serological tests are acceptable for ruling out type II HIT but lack still specificity and are also not standardised. 14C-SRA is time-consuming and is only available in highly specialized laboratories and not in all countries.

Aims:
The aim of our study is to develop a PMPs generation assay (PMPGA) in whole blood that could be routinely used to diagnose HIT. The ultimate goal is to provide a more specific functional test than the existing serological assays. In addition, this test should be easy to use,faster and as accurate as 14C-SRA.

Method:
The platelet-poor plasma of HIT-suspected patients (n=71) was first incubated 20 minutes at 37°C with citrated 109 mM whole blood from a healthy donor containing 0, 1 or 500 IU heparin/ml. Those three concentrations aimed at determining a basal PMP concentration, the PMP concentration generated by the HIT antibodies and checking the specificity, respectively. In the second step, PMPs positive or negative for annexin-V FITC (phosphatidylserine (PS)) and anti-CD62P-PE (P-selectin) were quantified on a BDIS FACS Aria® flow cytometer (BD Biosciences).The size of the PMPs was defined using a blend of monodisperse fluorescent beads (Megamix, BioCytex, Marseille, France) of three diameters (0.5, 0.9 and 3 μm) according to a previously described protocol. ROC Curves were performed to determine the optimal cut-offs for MPs concentrations and ratios. 4T's rule and clinical follow-up were registered. Results for ELISA, Light Transmission Aggregometry (LTA), PMPGA, and gold standard14C-Serotonin Release Assay (SRA) were compared by Chi-Square test.

Results:
During the incubation at low heparin concentration, PMPs expressing PS are generated due to the formation of immune complexes whereas PMPs rate decreases in presence of higher heparin concentration, allowing the definition of positive HIT. The determination of an optimal cut-off for MP concentrations requires standardization of many preanalytical and analytical conditions. However, ratios are much more convenient and the optimal cut-off for the ratio of PMP at low and high heparin concentration was calculated as 2. The relation between PMPGA and SRA was much more pronounced (p<0.0005, n=56 whose 9 positive SRA) than with LTA (p=0.0098, n=39) and ELISA (p=0.0374, n=53). In comparison to SRA, sensitivity and specificity of PMPGA were 78 and 98%, respectively. However, the 2 patients negative with PMPGA and positive with SRA were defined as HIT-negative afterwards. Indeed, the first patient had a 4T rule of 3 with negative serology and LTA. The second patient had a 4T rule of 6 with negative serology and positive LTA. She received low-molecular-weight heparin since more than 3 months and her thrombocytopenia increased after switch to danaparoid and hirudin. On preliminary data (n=19), circulating PMPs determination without heparin in the milieu allows positive and negative HIT plasma discrimination.

Conclusion:
PMPGA is a rapid and reliable test which reproduces the in vivo type II HIT reaction. It improves performances of LTA and is applicable in labs without LTA. It shows similar results than 14C-SRA with a slightly higher specificity. Finally, a screening test by circulating PMPs determination followed by a confirmation test with PMPGA could be an interesting and a simpler strategy improving HIT diagnosis.

Disclosures:
No relevant conflicts of interest to declare.