In Vitro Characteristics of Fondaparinux On HIT/HITT Samples.
Authors: Gould Nadia N , Haley Christine L , Song Lu , Sahud Mervyn A. , Dlott Jeffrey 
Published: October 2019

Abstract
Abstract 1319
Poster Board I-335
Heparin-induced thrombocytopenia (HIT) and HIT-associated thrombosis (HITT) are secondary to immune-mediated IgG antibodies that develop against the molecular complex of platelet factor 4 (PF4)–heparin, occurring with exposure to unfractionated heparin (UFH) and low molecular weight heparins (LMWH). Fondaparinux, a synthetic pentasaccharide that binds antithrombin and selectively inhibits Factor Xa, is indicated for prophylaxis and treatment of thrombosis. Fondaparinux has occasionally been reported to induce production of PF4–heparin antibodies; whether these antibodies activate platelets, in vitro or in vivo, remains unclear. Thus, fondaparinux has not been approved for patients with HIT/HITT. This study examined the in vitro cross-reactivity of fondaparinux in 2 HIT assays: a serotonin release assay (SRA) and an anti-PF4 enzyme-linked immunosorbent assay (ELISA) with neutralization. Serum samples (n=10) from patients with confirmed HIT/HITT were tested by SRA with UFH, LMWH (enoxaparin), and fondaparinux; and for anti-PF4 using a polyspecific ELISA (GTI, Waukesha, WI). In addition, 20 previously characterized samples strongly positive on the anti-PF4 ELISA (>1.000 OD) were used for confirmatory quenching studies with UFH, LMWH, and fondaparinux. LMWH-SRA and UFH-SRA results were concordant (positive) in all but one sample, which was positive with UFH but indeterminate with LMWH (Table). The fondaparinux-SRA was negative in 6 of 10 cases and indeterminate in 4. The indeterminate fondaparinux-SRA results were reproducible with a second platelet donor and when diluted 1:4 with negative anti-PF4 serum. Of note, platelet activation was observed in the buffer well (ie, without UFH, LMWH, or fondaparinux) in 3 of the 4 indeterminate cases but not in the 6 negative fondaparinux-SRA samples. All 10 samples showed positive results in the anti-PF4 ELISA (range=0.423-3.408 OD; median OD=2.588). In the second group of samples tested by anti-PF4 ELISA (range=1.395-3.204 OD; median=2.417 OD), quenching studies with excess UFH or LMWH yielded ≥50% inhibition, confirming anti-PF4 positivity, in 17 of 20 samples. Quenching with fondaparinux was not confirmatory, yielding only equivocal inhibition (range=0 to 46.7%; median=9.1%). In conclusion, fondaparinux appears to be generally not cross-reactive in SRAs performed on HIT/HITT-positive samples. The four fondaparinux-SRA cases were indeterminate despite heat inactivation of complement and the addition of hirudin to neutralize any thrombin contamination; this suggests that there may be heterogeneous pathogenic subpopulations that bind PF4 even in the absence of heparin or heparin-like molecules. The considerable variability in anti-PF4 confirmation studies with fondaparinux, relative to UFH and LMWH, warrants further study.
Table. SRA* and Anti-PF4 ELISA** Results from Patients with Clinical HIT/HITT UFH-SRA LMWH-SRA Fondaparinux-SRA Anti-PF4 ELISA Sample (% release) (% release) (% release) (OD units) 1 100 96 indeterminate 0.798 2 98 100 <20 2.573 3 34 indeterminate indeterminate 0.423 4 72 90 <20 3.012 5 100 107 indeterminate 3.275 6 95 92 <20 2.903 7 80 99 <20 2.602 8 74 70 <20 2.349 9 55 86 indeterminate 1.756 10 100 108 <20 3.408 * Positive result defined as ≥20% serotonin release with standard drug concentration and ≥50% inhibition with excess drug. Negative result defined as <20% serotonin release. Indeterminate result defined as ≥20% serotonin release with standard drug concentration and <50% inhibition with excess drug. ** Positive result defined as OD ≥0.400.

Disclosures
No relevant conflicts of interest to declare.