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 radioactive ¹⁴C 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 is expected to show >20% release of the ¹⁴C 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 is considered a better predictor of thrombosis than the anti-PF4 assay.

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 3.500 OD.

The anti-PF4 test has a sensitivity of >95% and a specificity ranging from 50% to 89%. 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.²

The combined sensitivity of the anti-PF4 and, the SRA is about 99%.³

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).⁵

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.⁶

Quest Diagnostics offers both tests individually, together as a panel, and as a reflex (a positive heparin-induced platelet antibody reflexes to SRA).

A cautionary note when ordering the panel or reflex—if the result interface (connection from Quest Diagnostics to your computer system) does not accept partial results, the anti-PF4 ELISA results will not be received until the SRA is reported. Confirming the status of your interface with an IT specialist is recommended. Alternatively, physicians may view all results irrespective of this issue using the Care 360 product.

Yes. Only IgG antibodies activate platelets, thus IgM and IgA classes are of minor clinical relevance.^7,8 Furthermore, the British Society for Haematology recommends that only the IgG isotype be tested.⁹

Quest Diagnostics does not perform the heparin neutralization procedure, sometimes referred to as a confirmatory step. It is our experience that virtually every positive sample is neutralized, and therefore we concluded that it offered 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 yielding a confirm negative result despite presence of clinical HIT/HITT.¹⁰

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

An SRA result is considered indeterminate when there is a >20% release of ¹⁴C serotonin in the patient serum/low dose heparin mixture and an absence of inhibition with high-dose unfractionated heparin (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.