Serotonin Release Assay
Functional HIT Assay
Heparin-dependent Platelet Antibody (Serotonin Release Assay)
Confirmatory assay used in the evaluation of heparin-induced thrombocytopenia (HIT)
Heparin-induced thrombocytopenia (HIT) is a potentially catastrophic, antibody-mediated complication of heparin therapy caused by immunization against platelet factor 4 complexed with heparin or other polyanions. HIT antibodies bind to PF4/heparin complexes on the platelet surface, resulting in platelet activation that leads to a platelet count decrease that can be accompanied by life-threatening thrombosis. This prothrombotic disorder can produce devastating thromboembolic complications, including ischemic limb necrosis, pulmonary embolism, myocardial infarction, and stroke.
Moderate thrombocytopenia is common in the clinical settings where heparin is administered and most cases are not caused by HIT. Differentiation of HIT from other potential causes of thrombocytopenia is the most difficult diagnostic component in the evaluation of heparinized, thrombocytopenic patients and relies on a combination of a clinical assessment and laboratory investigation. Prompt diagnosis and management is critical to minimizing the risk of life-threatening thrombosis. Patients diagnosed with or suspected of suffering HIT must be taken off heparin and transitioned to an alternative nonheparin anticoagulant as quickly as possible.
The laboratory investigation of HIT is challenging and requires correlation between clinical symptoms and laboratory assays. The most common assay performed is a serologic assay that detects the presence of HIT antibodies without regard to their functional ability. Several serologic assays that are relatively easy to perform are available commercially, and these are highly sensitive. The results of these assays have excellent negative predictive values and a negative result can be used to exclude HIT in all but the most compelling clinical circumstances. However, these assays suffer from low specificity and frequently yield positive results in the absence of clinical HIT. A positive result, especially of low titer, does not differentiate between pathogenic antibodies and clinically irrelevant antibodies. Functional assays that measure platelet activation by HIT antibodies in the presence of heparin are considered “gold standard” diagnostic laboratory tests due to their ability to detect the patient’s underlying procoagulable state in those with true HIT. Due to complexity of performance, functional assays that use washed-platelets are not widely available. The serotonin release assay (SRA) using washed-platelets is a well-established functional HIT assay that is both highly sensitive and specific for HIT.
Currently recommended HIT diagnostic algorithms published by the American Society for Hematology, British Committee for Standards in Haematology (BCSH) and American College of Chest Physicians Evidence-based Clinical Practice Guidelines incorporate an estimate of clinical probability and use of a sensitive immunoassay to guide initial management with subsequent confirmatory testing by a more specific functional assay.
Rarely, a spontaneous prothrombotic thrombocytopenia syndrome can occur without proximate heparin exposure. In this syndrome, the results of serologic tests and the serotonin release assay are indistinguishable from HIT, despite the fact that patients had not been treated with heparin. Warkentin and coworkers have proposed that a rigorous definition of spontaneous HIT syndrome should include otherwise unexplained thrombocytopenia/thrombosis without proximate heparin exposure and with anti-PF4/heparin antibodies that cause strong in vitro platelet activation—even in the absence of heparin.
The British Committee for Standards in Haematology (BCSH) 2012 published guideline states that “ideally, the diagnosis of HIT should be confirmed by a washed-platelet assay” such as the SRA.8 The 2012 CHEST Supplement on the diagnosis and treatment of HIT states “The washed platelet SRA and HIPA are generally accepted as the reference standard assays for HIT.”
Gel-barrier or red top tube
Transfer serum to a plastic transport tube. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.
Room temperature specimen received; grossly hemolyzed, icteric, and lipemic samples