A New Enzyme Immuno-Assay That Only Detects IgG Antibodies to Heparin-PF4 Complexes (Zymutest HIA IgG®) Improves the Specificity of the Diagnosis of HIT without Loss of Sensitivity.

Heparin-induced thrombocytopenia (HIT) is associated in most patients with the development of antibodies to heparin-modified platelet factor 4 (PF4). Commercial immuno-assays frequently detect these antibodies after cardiac surgery but only few patients develop clinical HIT. Therefore, platelet activation tests such as serotonin release assay (SRA) are necessary to ensure the diagnosis of HIT with high specificity. Another approach to increase diagnosis specificity could be to detect IgG antibodies which are of major clinical relevance since they are the only class able to directly activate platelets in the presence of heparin. Therefore, we evaluated the performances of a new commercial immuno-assay specific to IgG for the diagnosis of HIT Abs (Zymutest HIA IgG®, Hyphen Biomed, Neuville sur Oise, France). Samples from 101 patients with suspected HIT were analysed. 40 cases had developed significant levels of Abs to PF4 measured with PVS/PF4 ELISA (HAT45®,GTI, Brookfiled, WI, USA) and the diagnosis of HIT had been confirmed since SRA was positive. Every sample was then tested with a global assay named Zymutest HIA G/A/M® as followed: each diluted plasma (200 μl) was incubated for 1 hour with 50 μl of platelet lysate providing PF4 into wells previously coated with unfractionated heparin. After washings and incubation (1 hour) with anti IgG/A/M-HRP immunoconjugate, the enzyme activity was developed and absorbance was read at 450nm. In case of positive result (A₄₅₀ ≥ 0.5), the isotype distribution was analysed with a specific and standardized assay using monospecific anti-IgG-, anti-IgA- and anti IgM-HRP conjugates (Zymutest HIA-IgG® or -IgA® or -IgM®). A₄₅₀ values ≥ 0.5 were also considered as positive. GTI assay that detected IgG/A/M Abs to PVS/PF4 complexes in the 40 patients with HIT (Ss 100%) was also positive in 30 of the 61 cases with no HIT (Sp 50.8%). Comparatively, Zymutest HIA® global assay was positive in 39 of the 40 patients with HIT (Ss 97.5%) and in 14 of 61 cases without HIT (Sp 77%). On the other hand, significant levels of heparin-dependent IgG antibodies were also measured in these 39 HIT patients using Zymutest HIA IgG® assay (mean A₄₅₀: 1.81; range A₄₅₀: 0.5 – 2.76), and only in 6 patients without HIT (Sp: 90%). However, IgG levels measured in patients without HIT were significantly lower (mean A₄₅₀: 0.60; range A₄₅₀: 0.08 – 2.20) than in those with HIT (p < 0.0001). In addition, IgA or IgM heparin-dependent antibodies were only present, i.e. without IgG, in samples from patients for whom the diagnosis of HIT had been ruled out (n = 3). In conclusion, this study supports that the detection of significant levels of IgG heparin-dependent antibodies improves the diagnosis specificity in patients with a suspicion of HIT without loss of sensitivity. Nonetheless, whether IgG-specific immunoassays could avoid to perform platelet activation tests in patients with a strong pre-test probability of HIT warrant further study.