Variable serotonin release assay pattern and specificity of PF4-specific antibodies in HIT, and clinical relevance

Abstract

Background: The diagnosis of heparin-induced thrombocytopenia (HIT) requires functional assays to demonstrate that platelet factor 4 (PF4)-specific antibodies activate platelets, typically when therapeutic heparin (H) concentrations are tested ("classical" pattern). Some HIT samples also activate platelets without heparin ("atypical" pattern), but with unclear clinical significance.

Objectives: We aimed to assess whether platelet activation pattern and some characteristics of PF4-specific antibodies were associated with the severity of HIT.

Patients/methods: Serotonin release assay (SRA) pattern of 81 HIT patients were analyzed and compared with their clinical and biological data, including levels of anti-PF4/H immunoglobulin G (IgG) and anti-PF4 IgG in 47 of them.

Results: Higher anti-PF4/H IgG titers were measured in patients with an "atypical" SRA (optical density 2.52 vs. 1.94 in those with a "classical" pattern, p < .001). Patients of both groups had similar platelet count (PC) nadir and time to recovery, but those with an "atypical" SRA more frequently developed thrombotic events (69% vs. 34%, p = .037). Significant levels of anti-PF4 IgG were detected in both groups (38% and 61%, respectively). Whatever the SRA pattern, a lower PC nadir (35 vs. 53 G/L, p = .006) and a longer PC recovery time (6 vs. 3 days, p = .015) were evidenced in patients with anti-PF4 antibodies, compared with those with anti-PF4/H IgG only.

Conclusions: An atypical SRA pattern with elevated anti-PF4/H IgG titers seems associated with an increased risk of thrombosis in HIT. IgG antibodies to native PF4 may contribute to more severe and persistent thrombocytopenia, and their detection could be useful in clinical practice.

Keywords: antibody; platelet factor 4; platelets; serotonin release assay; thrombocytopenia; thrombosis.

FIGURE 1

Two types of SRA pattern were observed in our cohort of HIT patients and underlying mechanisms. (A, B) Platelet activation pattern of HIT patients with a classical SRA profile (no platelet activation without heparin, n = 34) or an atypical SRA profile (platelet activation without heparin, n = 13). Data are mean (%) ± SD. (C) Classical platelet activation pattern observed with the monoclonal anti‐PF4/H IgG antibody 5B9 tested at low concentrations (10 and 20 μg/ml) without and with increasing concentrations of heparin. (D) Three mechanisms contributing to an atypical platelet activation in SRA: high concentrations of anti‐PF4/H IgG, as demonstrated with the monoclonal anti‐PF4/H IgG 5B9 tested at 200 μg/ml; high PF4 concentrations in platelets environment, as shown with 10 μg/ml of exogenous PF4 added to 5B9 20 μg/ml; or the presence of anti‐PF4 IgG antibodies, illustrated here with the monoclonal anti‐PF4 IgG 1E12 at 2.5 μg/ml. In (C, D) data are given as mean ± SEM of n = 3 or 4 experiments. HIT, heparin‐induced thrombocytopenia; SRA, serotonin release assay.

FIGURE 2

Comparison of anti‐PF4/H IgG titers, plasma PF4 concentrations and anti‐PF4 IgG titers in patients with a classical or an atypical SRA pattern in our cohort of HIT patients. Assessment of anti‐PF4/H IgG titers (A), plasma PF4 concentrations (B), anti‐PF4 IgG titers (C) in HIT patients according to their platelet activation pattern without heparin in SRA (classical profile: no platelet activation, n = 34; atypical profile: platelet activation, n = 13). Each circle represents a HIT patient, and the solid line indicates the median value of data obtained. Dashed lines in (A and C) represent the cutoff values of each assay. HIT, heparin‐induced thrombocytopenia; SRA, serotonin release assay

FIGURE 3

Platelet count nadir in heparin‐induced thrombocytopenia (HIT) patients according to the presence of anti‐PF4 IgG antibodies. Platelet count nadir in patients without (n = 26) or with (n = 21) detectable anti‐PF4 IgG antibodies. Each circle indicates a HIT patient. The table indicate the proportion of patients (n, %) with platelet count nadir below and ≥50 G/L.