Immunoassays for heparin-induced thrombocytopenia (HIT)

There are 2 major categories of immunoassays for anti-PF4/heparin (H) antibodies (also called PF4-dependent immunoassays):

  1. Enzyme immunoassays (EIAs):
    • Advantages:
      • High sensitivity (98–99%) for detecting pathological (platelet-activating) HIT antibodies.
      • Semi-quantitative – higher optical densities predict greater likelihood of HIT.
    • Disadvantages:
      • Lower specificity compared to functional assays such as serotonin release assay.
      • Testing occurs in batches, which may delay results for 24 hours or longer.
  2. Latex immunoturbidimetric assay:
    • Advantages:
      • Possibly higher specificity compared to EIAs.
      • High sensitivity (about 98%).
      • Semi-quantitative – higher result predicts greater likelihood of HIT.
      • Rapid, on-demand, fully-automated laboratory assay (run on standard coagulation instrument).
      • Once the sample is in the lab a result can be made available in 30 minutes or less.

In both cases, the assay detects antibodies directed against the anti-PF4/heparin complex.

Enzyme immunoassay (EIA)

Antibodies (HIT-IgG) from patient plasma bind to the PF4-heparin complexes on the microtiter plate. Multiple washes are performed here and after all subsequent steps. Alkaline-phosphatase conjugated antibodies are then added, which recognize and bind to the patient’s HIT antibodies. A substrate for the alkaline phosphatase enzyme is added to the reaction mixture, and this results in a color change, the strength of which is proportional to the quantity of antibodies bound to the PF4-heparin complexes. The change may be quantitated using a spectrometer, and the results are reported as optical density.

Latex immunoturbidimetric assay (LIA)

LIA is a “functionalized immunoassay” that detects HIT antibodies based on their ability to competitively inhibit agglutination of HIT-like antibody-bearing particles. The readout is light transmission. Normally, the particles agglutinate when mixed with complexes of PF4-heparin surrogate, which results in higher absorbance (lower light transmission). Addition of patient plasma containing PF4/heparin-reactive antibodies (HIT-IgG) inhibition of particle agglutination, resulting in decreased absorbance and higher light transmission. Although the LIA is classified as an immunoassay, it differs from most other immunoassays, such as EIAs, in that the presence of PF4/heparin-reactive antibodies within patient plasma results in inhibition of particle agglutination through competition with a HIT-mimicking monoclonal antibody.