Vol. 15 •Issue 7 • Page 115
Laboratory Technology HIT Assessment
Heparin is one of the most widely prescribed medications in the United States, with an estimated 12 million patients receiving the drug annually.1 Although the most common adverse event associated with heparin is bleeding, a significant number of patients develop a prothrombotic state known as Heparin-Induced Thrombocytopenia (HIT), the clinical course of which can be as mild as an asymptomatic decrease in platelet count or as severe and potentially devastating as a thromboembolic complication.
Type I or Heparin-Associated Thrombocytopenia (HAT) is mild and transient, and usually not associated with thrombosis. Normally, there is no need to stop heparin therapy, since platelet counts will usually return to normal even if therapy is continued.2 Type II, commonly referred to as HIT, is an immune-mediated condition that occurs in approximately 1 percent to 5 percent of patients receiving heparin therapy. Typically, HIT occurs between day five and 10 after initiation of Heparin therapy, but patients may present with clinical symptoms within minutes or hours of a Heparin rechallenge if the prior exposure was within the previous 100 days.3 There also have been reports of delayed-onset HIT that begin several days to weeks after heparin has been discontinued.4,5
The major determinant in the pathogenesis of HIT appears to be antibodies to the Heparin/Platelet Factor-4 (HPF4) complex.6-8 These antibodies are most frequently induced by Unfractionated Heparin (UFH) use following cardiopulmonary bypass surgery and major orthopedic surgery.9 Although HIT is more common in patients receiving large doses of heparin therapy intravenously, HIT can occur following incidental exposure to heparin through Heparin flushes, subcutaneous administration or Heparin-coated catheters and prostheses, such as those used in chronic dialysis patients.10
Functional, Antigen Assays
Two main categories of laboratory assays can assist in the clinical diagnosis of HIT: functional and antigen assays. Functional or activation assays utilize donor platelets to measure the platelet-activating potential of HPF4-antibody complexes in the presence of therapeutic concentrations of heparin. These CLIA-classified high complexity methods include the Serotonin-Release Assay (SRA), Platelet Aggregation Test (PAT) and Heparin-Induced Platelet Activation Assay (HIPA). Although these methods have a high probability of identifying cases clinically diagnosed as HIT positive, the variability of donor platelets is a major factor influencing test sensitivity.11-13 In addition, functional assays are technically demanding, very time consuming and often outsourced to reference laboratories. Institutions may experience delays in obtaining results and incur costly fees to obtain a single patient determination.11,14
Antigen assays, also known as immunoassays, detect antibodies of the three major immunoglobulin classes (IgG, IgM, IgA) against Platelet Factor-4 (PF4) bound to heparin or a heparin substitute.11,15 Although antigen assays do not measure the ability of antibodies to cause platelet activation, a functional response associated with HIT, when performed on specimens obtained from patients clinically suspected of having the complication, these screening tests have a high sensitivity for diagnosing HIT.14,16
Until recently, the enzyme-linked immunoassay (ELISA) was the only antigen assay available. ELISAs are most efficiently run in batch, often at a maximum of once daily depending on the institution.
Providing Qualitative Determination
In 2005, Akers Biosciences Inc. launched the PIFA Heparin PF4 Rapid Assay, a unit-use device that is CLIA-classified as moderate complexity. The rapid manual assay provides clinicians with a qualitative determination of the presence or absence of HPF4 antibodies.
The procedure consists of five steps once a patient’s 20 µL fresh serum specimen is introduced into the device. The test is based on Akers’ proprietary Particle ImmunoFiltration Assay (PIFA) platform whereby a patient’s serum is put into the device and mixes with the reagent system. Blue microparticles coated with purified PF4 protein form the basis of the reagent system, and enhancing agents promote rapid matrix formation of these microparticles in the presence of HPF4 antibodies. Visual color results are produced through the interaction of these microparticles with a membrane-filtration system. Nonreactive, monodispersed blue microparticles will freely migrate through this membrane system and become visible in the test result window, indicating a negative result.
The intensity of the blue color in the test result window will vary as the rate reaction and aggregation of microparticles differ among samples. Any trace of blue in the test window (ranging from a darker, bright blue to a very faint tint of blue) along with a red color in the control window is considered a negative result.
Reactive, matrixed microparticles will be trapped by the membrane system and no blue color will be visible in the test result window, indicating a positive test result. The white-to-pale grey color in the test window represents one example of a positive result; the test result circle may also take on the color of the patient’s serum, but no blue color will be present.
The PIFA Heparin PF4 Rapid Assay can provide clinicians with a means of including Heparin/PF4 antibody status in an assessment algorithm for HIT.
Dr. Seckinger is a practicing pathologist, laboratory director and director of Clinical Development for Akers Biosciences Inc.
