Background: Laboratory confirmation of heparin-induced thrombocytopenia (HIT) relies on functional assays highlighting the presence of heparin-dependent platelet-activating antibodies in the plasma or serum of patients. Functional assays using washed platelets, i.e., ¹⁴C-serotonin release assay and heparin-induced platelet activation assay (HIPA) are considered gold standards because of their excellent specificity (> 99%) and good sensitivity (> 95%). However, several recent data suggest that the addition of exogenous platelet factor 4 (PF4) in functional assays using washed platelets could improve their ability to detect low levels of pathogenic HIT antibodies.

Aims: To investigate whether the addition of PF4 enables to detect the presence of low levels of platelet-activating antibodies in the plasma of patients investigated for HIT that failed to cause platelet activation in the conventional HIPA test.

Methods: Plasma samples from 28 patients with suspected HIT were studied. The presence of anti-PF4/heparin antibodies was evaluated with an immuno-assay specific to IgG (Zymutest HIA IgG, Hyphen Biomed). HIPA was performed as previously described and after adding 10 µg/ml of purified human PF4 (Hyphen Biomed) in the reaction mixture (HIPA-PF4).

Results: HIPA-PF4 detected platelet-activating antibodies in 11 (100%) of the 11 HIPA-positive plasma (HIPA^pos). Platelet-activating antibodies were detected in 5 (29%) of 17 samples that were positive for the immuno-assay but tested negative or undetermined in the HIPA (HIPA-PF4^pos). None of the HIPA-PF4 negative plasma was positive in the conventional HIPA (HIPA^neg, Table 1). Importantly, the addition of PF4 in HIPA allowed to detect significantly lower levels of platelet activating PF4-specific IgG than conventional HIPA (mean optical density = 1493 ± 192 vs 1961 ± 463, Mann Whitney p=0.04, Figure 1).

Conclusions: Addition of exogenous PF4 might improve the detection of weakly activating HIT antibodies by HIPA and make the test more sensitive.

[Table 1]

[Figure 1. Levels of anti-PF4/heparin IgG and reactivity in the HIPA-PF4]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/addition-of-exogenous-platelet-factor-4-in-the-heparin-induced-platelet-activation-hipa-assay-for-the-detection-of-pathogenic-heparin-induced-thrombocytopenia-antibodies/