Anti-PF4 and Serotonin Release Assay (SRA) for Diagnosing Heparin-induced Thrombocytopenia/Thrombosis (HIT/HITT)

The anti-PF4 (ie, heparin-induced platelet antibody) test is an immunologic, enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the platelet factor 4 (PF4)/heparin complex. The assay uses heparin bound to protamine sulfate, and PF4 is supplemented through a platelet lysate.

The SRA test is a functional assay that measures heparin-dependent platelet activation. Patient serum is incubated with donor platelets containing radiolabeled serotonin and different concentrations of heparin. Antibody present in the patient serum will bind and activate donor platelets, releasing radiolabeled serotonin from the platelet granules. A positive SRA test is expected to show >20% release of the  radiolabeled serotonin when mixed with patient serum and low-dose heparin. In addition, the high-dose heparin must reduce the % release obtained with low-dose heparin by at least 50% in order to demonstrate that the platelet activation is heparin-dependent.

The SRA test is considered a better predictor of thrombosis than the anti-PF4 test.¹

OD is the optical density; it is an indication of antibody strength. Antibody present in the patient sample binds to the heparin-PF4 coated wells, leading to a color-producing reaction. A higher antibody concentration leads to more color production and a higher OD reading. The positive cutoff is ≥0.300 OD, and high-titer antibodies may yield values up to 3.000 OD. 

The anti-PF4 test has a sensitivity of ~99% and a specificity ranging from 30% to 70%.² However, the higher the OD, the greater the probability is for clinical HIT/HITT. Similarly, the specificity increases when the OD is >1.000.¹

The SRA test has a sensitivity of 88% to 100% and a specificity of 89% to 100% for HIT.³

The combined sensitivity of the anti-PF4 and the SRA tests is about 99%.¹

No, testing for suspected HIT is not needed in this situation. HIT can be excluded based on a low pretest probability score (4T’s score of 0-3).⁶

A published algorithm that incorporates the clinical assessment suggests that patients with a moderate pretest probability and the laboratory results above are “indeterminate” for HIT/HITT.⁴

This scenario may represent a true antibody response, but one that lacks sufficient titer or avidity to activate platelets in vitro. The fact that the antibodies are measurable only with the anti-PF4 ELISA may also be indicative of the assay’s greater sensitivity. While this explanation has a good scientific foundation, some patients may still have clinically significant antibodies, thereby the rationale for the “indeterminate for HIT/HITT diagnosis.” False-positive anti-PF4 ELISA results have also been attributed to cross-reactivity to antiphospholipid antibodies.⁷

Yes. Only IgG antibodies activate platelets, thus IgM and IgA classes are of minor clinical relevance.^8,9 Furthermore, the British Society for Haematology recommends that only the IgG isotype be tested.¹⁰

Quest Diagnostics does not perform the heparin neutralization procedure, sometimes referred to as a confirmatory step. In Quest’s experience, virtually every positive sample is neutralized, and therefore offers no significant insight beyond that of the OD value. A retrospective study also reported that some patients with high ELISA OD values were misclassified by a confirmed negative result despite presence of clinical HIT/HITT.¹¹

Given the short half-life (30 minutes to 2 hours) of unfractionated heparin (UFH), waiting a minimum of 2 hours is suggested. 

A SRA result is considered indeterminate when there is a >20% release of radiolabeled serotonin in the patient serum/low dose heparin mixture and an absence of inhibition with high-dose UFH. There may be other nonheparin-dependent antibodies present (ie, Class I HLA antibodies or platelet receptor specific antibodies) that may cause platelet activation.

A low-positive SRA is repeated using platelets from a different donor to demonstrate reproducibility. At this point, specific conclusions about a low-positive result cannot be made. It is recommended that the result be considered in the context of all the data at hand, specifically the clinical assessment and anti-PF4 result.