In Vitro Characteristics of Fondaparinux On HIT/HITT Samples.

Abstract

Abstract 1319Poster Board I-335Heparin-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.TableSRA* and Anti-PF4 ELISA** Results from Patients with Clinical HIT/HITTUFH-SRALMWH-SRAFondaparinux-SRAAnti-PF4 ELISASample(% release)(% release)(% release)(OD units)110096indeterminate0.798298100<202.573334indeterminateindeterminate0.42347290<203.0125100107indeterminate3.27569592<202.90378099<202.60287470<202.34995586indeterminate1.75610100108<203.408*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. DisclosuresNo relevant conflicts of interest to declare.