Saline whole-blood impedance aggregometry for detection of heparin-independent platelet-activating anti-platelet factor 4 antibodies.

Abstract

This study evaluates the practice of incorporating a saline testing condition into the heparin-induced thrombocytopenia (HIT) functional assay using whole-blood impedance aggregometry (WBIA) to identify a subgroup of highly pathogenic anti-platelet factor 4 (PF4) antibodies with heparin-independent platelet-activating properties. From 2023 to 2025, we analyzed 150 evaluable patients (65 WBIA Negative, 85 WBIA Positive). Positive cases were subdivided into Classic-Positive (low-dose heparin positive only; n = 46) and Saline-Positive (positive under both low-dose heparin and saline; n = 35). The diagnosis of clinical HIT by hematologists was used as reference. PF4/IgG was measured by chemiluminescent immunoassay; WBIA was performed with low-dose heparin, high-dose heparin, and saline; and available serotonin release assay (SRA) results were analyzed. Saline-Positive patients exhibited significantly higher incidences of HIT-associated thrombosis (89% vs 54%, P = .005), delayed-onset HIT (23% vs 0%, P = .001), refractory HIT (40% vs 7%, P < .001), spontaneous HIT (14% vs 0%, P = .013), and more venous thromboembolism (pulmonary embolism and deep vein thrombosis, P < .05) compared with Classic-Positive patients. All 35 Saline-Positive cases were classified as autoimmune HIT (aHIT). The spontaneous HIT subset (n = 5) displayed a distinct pattern of low-titer anti-PF4 antibodies discordant with strong saline-WBIA reactivity and a high SRA false-negative rate (57.1%). High-dose intravenous immunoglobulin neutralized saline positivity in real time. Saline-positivity in WBIA identifies a heparin-independent aHIT phenotype with higher pathogenicity and thrombotic risk. This rapid saline protocol addresses diagnostic gaps where standard 2-condition SRA protocols may fail, offering a practical tool to identify high-pathogenic anti-PF4 antibodies and guide clinical management.