The Impact of Non-H:PF4 Antibodies on Results of the Serotonin Release Assay for Heparin-Induced Thrombocytopenia.

Background: The Serotonin Release Assay (SRA) is frequently used to test for the presence of heparin-platelet factor 4 (H:PF4) antibodies as part of a diagnosis of Heparin-Induced Thrombocytopenia (HIT). In this test, immune complexes formed when antibodies bind to soluble or membrane-bound H:PF4 complexes, activate platelets via FcγIIa receptors. A HIT-positive SRA demonstrates donor platelet activation when test sera are incubated with low concentrations (0.1–1.0 U/ml) of heparin, but not when the incubation includes excess, neutralizing levels (10–100 U/ml) of heparin. Platelet activation at both low and high heparin is designated an indeterminate result. This heparin-independent platelet activation can be caused by immune complexes unrelated to HIT, by other anti-platelet antibodies or by platelet agonists. The present studies were conducted to study the occurrence of anti-platelet antibodies in specimens with marginal or inconclusive SRA activity.

Methods: The study included two populations of specimens that had been previously tested by both SRA and H:PF4 antibody ELISA (GTI, Brookfield, WI). Group I included 44 specimens that tested SRA positive in spite of the absence of measurable H:PF4 antibodies. Most were relatively weak in the SRA: 51% ± 3.4 % (S.E.) serotonin release with 0.1 U/ml heparin. Group II consisted of 18 specimens that gave an indeterminate SRA response: heparin-independent platelet activation. Of these, 5 were positive for H:PF4 antibodies and 13 were negative. All specimens were analysed by PakPlus ELISA screening (GTI, Brookfield, WI) to determine if antibodies to HLA Class I and/or to common platelet specific glycoproteins (GPs) were present.

Results: In Group I, 19 of the 44 (43%) specimens tested positive for one or more anti-platelet antibody. 18 of the 19 (95%) had either anti-GP IIb/IIIa (n=10)(53%) and/or anti- HLA Class I (n=11)(58%) antibodies. One specimen had antibodies to GP Ib/IX and to GP IV. The remaining 25 (57%) specimens tested negative. In Group II, 13 of the 18 (72%) SRA-indeterminate specimens had detectable anti-platelet antibodies. All but one of the 13 (92%) included antibody to HLA Class I. The anti-glycoprotein antibodies were less frequent in this group: anti-GP IIb/IIIa (n=2)(15%), GP Ia/IIa (n=3)(23%), GP Ib/IX (n=2)(15%) or GP IV (n=3)(23%).

Conclusion: Non-H:PF4 anti-platelet antibodies, especially antibodies to GP IIb/IIIa or to HLA Class I, are not uncommon in sera referred for SRA testing. Specimens containing anti-platelet antibodies can give a positive or an indeterminate response in the two-point SRA. Specimens without H:PF4 antibodies that test positive in the SRA should be scrutinized for anti-platelet antibodies as an alternative to the diagnosis of HIT. Also, an indeterminate SRA should not be considered a negative test result. Anti-platelet antibodies alone can cause a non-heparin dependent platelet activation: however, their presence may also mask a positive, heparin-dependent, SRA response to H:PF4 antibodies. Finally, it is not uncommon for specimens to test positive by the 2-point SRA in the absence of antibodies to H:PF4 or to other platelet antigens. The mechanism for this response, and its significance to diagnosis of HIT, requires further investigation.