Table 2. Immunologic and Functional HIT Assays.

Test	Purpose	Method	Reference range
Heparin-platelet factor 4 (PF4) enzyme-linked immunosorbent assay (ELISA)	Immunologic HIT assay that measures the amount of HIT antibodies directed against PF4 (detects functional and nonfunctional antibodies)	Patient serum is added to a microtiter plate coated with PF4 (or heparin-PF4 complex) Alkaline-phosphatase linked secondary anti–human IgG antibody is added and incubated with the test samples After washing, a colorimetric substrate is added and the optical density of the colored product is measured after a 30-min incubation	Optical density values 0-0.499 (of note, each integer unit increase equals a 10-fold increase in signal intensity)
Serotonin release assay (SRA)	Functional HIT assay that measures serotonin release from dense granules in platelets as a marker for platelet activation in the presence of high and low doses of heparin	Patient serum is added to donor platelets labeled with 14C-serotonin in the presence of low (0.1 U/mL) and high (100 U/mL) concentrations of heparin (and in some specialized reference laboratories, buffer control) Platelet activation is determined by the amount of 14C-serotonin released into the reaction supernatant	<20% Platelet activation difference between the low and high concentrations of heparin Saline buffer is not generally tested; if performed, there will not be significant serotonin release in buffer for classic HIT
Latex immunoturbidimetric assay (LIA)	Functional immunologic HIT assay that detects the presence of PF4 HIT antibodies based on their ability to competitively inhibit agglutination of HIT-like monoclonal antibodies bound to latex particles	Patient plasma is added to latex particles coated with a HIT-like monoclonal antibody and PF4 molecules conjugated with fluorescent marker With negative plasma (those without HIT antibodies), the latex/monoclonal antibody particles and PF4 molecules agglutinate, which will increase detectable fluorescence Positive plasma will compete with the latex/monoclonal antibody particles leading to less agglutination and lower fluorescence	≥1.0 U/mL
p-Selectin expression assay (PEA)	Functional HIT assay that measures platelet surface p-selectin expression as a marker of platelet activation in the presence and absence of high-dose heparin	Patient serum is incubated with platelets pretreated with PF4 purified from normal donor platelets ± high (100 U/mL) concentration of heparin Platelet activation is determined by measuring the presence of p-selectin (CD62p) by flow cytometry	Percent release with PF4 vs PF4 + high-dose heparin, reported as positive, negative, or borderline

Abbreviation: HIT, heparin-induced thrombocytopenia.