A New Rapid Test for the Detection of PF4:Heparin Antibodies.

Abstract 3708

Heparin Induced Thrombocytopenia (HIT), the most frequent drug-induced immune-mediated type of thrombocytopenia, is an important and sometimes life-threatening complication of heparin therapy. HIT can be triggered by standard therapeutic dose heparin, low-dose (prophylactic treatment), low molecular weight heparin, unfractionated heparin and even by minute quantities given to flush intravascular catheters.

HIT is a clinicopathologic syndrome cause by platelet activating antibodies that recognize complexes of platelet factor 4 and heparin (PF4-heparin). Thrombocytopenia occurring within 5 – 10 days of administration of heparin is the most common clinical effect. The most severe complication of HIT is the occurrence of both venous and arterial thrombosis. Venous thrombosis can result in limb gangrene and the need for limb amputation.

The diagnosis of HIT is based on clinical abnormalities including thrombocytopenia with or without thrombosis and the detection of antibodies to the PF4-heparin complex (termed PF4 dependant antibodies). There are two major types of assays for the detection of heparin dependant antibodies: functional assays and PF4 dependant antigen immunoassays. Functional assays include the serotonin release assay (SRA) and the heparin induced platelet activation assay (HIPA). Enzyme linked immunoassays (ELISAs) are the most frequently used PF4 dependent antigen immunoassay. Functional assays are technically difficult to perform and are considered to be complex specialty assays. ELISAs while not complex are not generally available on a STAT basis. There is a need for a simpler assay which retains or improves the sensitivity and specificity of the existing immunoassays that can be available on a STAT basis.

GTI Diagnostics is developing a rapid test based on the proprietary technology used in GTI's PF4 Enhanced® ELISA. The PF4 Rapid test is a single use, semi-quantitative assay contained in a cartridge. It is designed to detect anti-PF4-heparin antibodies in human serum and/or plasma with a total assay time of 15 minutes. The assay uses only 5 μL of human serum and/or plasma along with a proprietary antigen and a proprietary fluorescent secondary antibody used for detection. The assay results are read using a fluorescent reader. A method comparison study was conducted which included an evaluation of 83 serum samples obtained from patients receiving heparin of which 36 were positive and 22 were negative in the PF4 Enhanced® assay. These samples were also tested in the PF4 Rapid Test assay. In addition 25 samples from normal individuals who were not receiving heparin were tested in both assays. Analysis of the data using a 2×2 table (shown below) resulted in a sensitivity of 100%, a specificity of 93.6% and an overall agreement of 96.4% of the PF4 Rapid Test compared to the PF4 Enhanced®.

Although HIT is a true clinicopathological syndrome, its diagnosis still rests primarily on clinical grounds since laboratory tests may not be available locally or may not be available in a sufficiently timely manner. Owing to the high risk of thrombosis associated with HIT, antithrombotic therapy with alternative anticoagulants should be started immediately when serologic assays confirm clinical suspicion. Readily available results, obtained from immunoassays for rapid detection of antibodies against PF4-heparin complexes, may be combined with the pretest estimation of clinical probability in order to exclude and/or confirm the diagnosis of HIT; therefore, assisting physicians in the time-sensitive clinical management of HIT.