Simon Davidson MPhil FIBMS CSci
Department of Haematology,
Royal Brompton & Harefield NHS
Heparin-induced thrombocytopenia (HIT) is a life-threatening adverse drug reaction to heparin that must be quickly diagnosed, allowing for appropriate alternative anticoagulant strategies to be employed. HIT is characterised by a rapid fall in the platelet count 5–10 days after starting heparin therapy with or without thromboembolic complications and most commonly seen in cardiac and orthopaedic patient populations. The thrombocytopenia is caused by antibodies directed against platelet Factor 4 (PF4)/heparin complexes. Many methods for anti-PF4/heparin detection exist such as enzyme immunoassays (EIA), which have been shown to exhibit high negative predictive value (NPV) allowing for the exclusion of HIT in the majority of suspected patients; however, most EIAs are performed in a batches, thereby delaying results to the physician. The highly specific functional assays require either a known HIT antibody reacting blood donors platelets or panel of a minimum of four to six blood donors’ platelets to perform the tests. The 4Ts scoring system(1) can be used to estimate the clinical probability of HIT in a patient but, once a moderate or high 4T score is obtained in a patient where the spectre of HIT has been raised, a confirmatory test is generally then required to rule HIT in or out. The timing of this test result is extremely important as if there are no obvious clinical manifestations of HIT, thromboembolic disease, the decision of whether to continue heparin, inexpensive and easy to monitor or switch to a more costly alternative that is invariably more difficult to monitor becomes key. Alternative anticoagulant options are also diminishing, with lepirudin (Refludan) being withdrawn from the market in April 2012 and a perception that there is an epidemic of overdiagnosis of HIT putting many patients at an unnecessary risk.(2) There is a need therefore for rapid immunological assays that will detect the PF4/heparin antibodies that are clinically significant allowing alternative anticoagulant therapy to be started.
Heparin has been known since the early 1900s and the first clinical trials of its use as an anticoagulant for venous thromboembolism took place in the mid-1930s. It remains a widely used anticoagulant for the prophylaxis or treatment of thrombotic diseases. HIT has been recognised for 50 years but it was not until the 1990s that the target antigen was identified.(3) HIT is an immunological severe adverse drug reaction to heparin therapy that can be fatal if undiagnosed and known to be associated with both unfractionated (UFH) and low molecular weight (LMWH) heparin. HIT is one of the most common of all adverse drug effects owing to the volume of patients receiving heparin therapy. If untreated, risk for thrombosis and subsequent morbidity and/or mortality increases significantly. The difficulty in treating HIT begins with the difficulty in diagnosis. Two well-recognised guidelines exist for the diagnosis, treatment, and management of HIT suspected patients: Treatment and Prevention of Heparin-Induced Thrombocytopenia. American College Of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th edition)(4) and The Management of Heparin-Induced Thrombocytopenia by the British Committee for Standards in Haematology.(5) Both guidelines acknowledge that HIT is a clinicopathologic finding and that laboratory testing is only adjunct to clinical impression, though integral to final diagnosis. Both guidelines also recognise the difficulty many laboratories have in providing timely reporting of clinically sensitive and specific results. The testing algorithms suggested in these guidelines as well as those widely proposed in publications attempt to support the varying levels of PF4/heparin antibody testing offered within the hospital laboratory environment. The presence of PF4/heparin antibodies can be detected using platelet activation assays and immunological assays. The pathoclinically significant antibodies are those of the IgG subclass and it is generally thought a test that will only detect these antibodies is therefore much more specific in identifying the clinically significant HIT patients. In the cardiac population the appearance of HIT antibodies is very common and ~50% of patients treated with heparin will develop PF4/heparin antibodies.(6) However >98% of these are non-clinically significant antibodies (IgA/M) that do not cause the pathological features of HIT. A rapid specific antibody test is therefore highly desirable.
It has been shown that the diagnostic specificity of the platelet activation assays is greater than the immunoassays; however, testing often requires referral of patient blood samples to specialised laboratories, thereby increasing the decision making time allowed to the clinician. In some cases, HIT is assumed without the confirmation of laboratory results in an effort to reduce turnaround time for patient management, leading to unnecessary therapeutic changes. Alternative anticoagulants may pose a patient management challenge; increase bleeding risk; present a difficult transition to warfarin; or, increase drug cost. Excluding HIT in suspected patients may prevent unnecessary alternative anticoagulant treatment. Alternatively, it is recommended that non-expert laboratories should use an immunoassay that detects IgG antibodies against PF4/polyanion complexes of high sensitivity primarily to rule-out HIT.(7) In general, the negative predictive value of PF4/heparin antibody assays is high, while the clinical relevance of the detection of PF4/heparin antibodies strongly depends on the clinical context.
Widely accepted commercial immunoassays can be easily used by scientists in a clinic laboratory; most methods take two hours to perform (Technoclone HIT IgG ELISA only takes 70 minutes) and testing in a batch method is often prefered. Rapid results from on-demand testing can aid in the exclusion or confirmation of HIT and help direct appropriate therapeutic decisions. This can be facilitated by use of an on-demand model for HIT antibody testing. Such a model would include pre-testing clinical assessment completed by the physician and determination of PF4/heparin antibody presence and level as provided by the laboratory. Immunoassays to detect PF4/heparin antibodies are the usual first-line tests. A negative antigen assay combined with low or moderate pre-test probability has good negative predictive value, making a diagnosis of HIT unlikely and the exclusion of HIT possible. A strong-positive antigen assay result in a high-probability patient is strongly suggestive of HIT.
Commercially available assays
There are five commercial ELISA assays available to detect either IgG only or IgG/A/M antibodies. They vary in the way PF4 is made available in the assay, for example, surface-bound PF4-heparin (Asserachrom HPIA, Diagnostica Stago, Asnières, France) or polyvinylsulphate-PF4 surface bound (GTI-PF4, Quest, Knowle, UK). One ELISA assay variation is to use heparin bound to a solid phase (Zymutest, Hyphen, Quadratech, Surrey, UK) this allows heparin complexes and other chemokines that exhibit heparin affinity to bind to the functionally active heparin. The following commercial companies all produce IgG only ELISA assays, STAGO, Technoclone, AESKU diagnostics, Hyphen and GTI. All assays take approximately two hours to perform, except the Technoclone assay, which takes 70 minutes. If positive, the ELISA can be repeated using high-dose heparin (100 U/ml). Inhibition of a positive result by more than 50% reduction in the optical density is characteristic of clinically significant HIT antibodies.(8) Although positive/negative cut-off values are provided by the manufacturers, the negative absorbance cut-off for the ELISA should be established locally using mean ± 3 standard deviations.
There are rapid screening tests available detecting IgG/A/M class antibodies, some of which are automated (Chemiluminescent HIT screen IgGAM and IgG specific and IgG/A/M immunoturbidometric assay, Hemosil, Instrumentation Laboratory, Cheshire, UK). HemosIL HIT-Ab(PF4-H) is one of the new, rapid methods for the detection of total immunoglobulin against PF4/H complexes on ACL TOP family of coagulometers.(9) It uses a competitive agglutination method for PF4-polyninyl sulphate complexes with citrated plasma and results are available within 13 minutes. Using a cut-off value of 1.0U/mL for HemosIL HIT-Ab(PF4-H), the new test was compared with Asserachrom HPIA ELISA screening test IgG/A/M HIT antibodies. Results showed a matching positivity of 60.2% (95%CI: 48.9–70.8%), matching negativity of 94.6% (95%CI: 91.5–96.7%), and the overall agreement of 87.7% (95%CI: 84.1–90.7%). These results are comparable to other PF4/H antibody assays available; with the added benefit of full automation and on-demand testing which provides results at the critical moment when physicians are required to make clinical decisions regarding anticoagulant therapy.
Other rapid screening tests include the gel particle agglutination method (Diamed, Midlothian, UK), using polymer particles coated with heparin/PF4 that act as the solid phase, or the PIFA Heparin/PF4 mini reactor (Akers Biosciences, Quadratech, Surrey, UK). All of these assays provide rapid results, <30 minutes, are highly sensitive but lack specificity. These assays also have positive and negative controls or a control built into the test.
A new nanoparticle-based lateral flow immunoassay has just been launched by STAGO.(10) This is a single test strip with built in control and provides results within 10 minutes. It detects IgG antibodies against PF4/polyanion complexes. A small volume of patient serum (5µl) is added to the test strip along with a buffer containing ligand-labelled human PF4 complexed to a polyanion. As the buffer and sample travel through the strip the PF/polyanion complexes bind to a gold nanoparticle coated with the anti-ligand and at the same time any human antibodies against PF/polyanion complexes also bind to this super complex. When the fluid mixture reaches the location of the test line within the strip any complexes containing the gold nanoparticle, PF4/polyanion and human antibodies are prevented from progressing by an immobilised goat antibody to human IgG. Initial results have shown this to have very good performance characteristics when compared to established EIA assays.
The immunological tests for HIT generally have high sensitivity, usually 80–100%, for the heparin/PF4 antibodies but the specificity is low; however, a strongly positive test indicates a much greater probability of HIT than a weakly positive test. A higher ELISA optical density measurement has also been significantly correlated with thrombosis.
The diagnosis of HIT remains a challenge but with the use of a clinical scoring system accompanied by a rapid immunological screening test for HIT antibodies, an improvement in diagnosis may be possible, thereby helping clinicians to quickly diagnose this life-threatening condition and avoiding inappropriate changes of anticoagulation treatment in patients that do not have HIT.
- Lo GK et al. Evaluation of pretest clinical score (4 T’s) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings. J Thromb Haemost. 2006;4:759–65.
- Cuker A. Heparin-induced thrombocytopenia (HIT) in 2011: An epidemic of overdiagnosis. Thromb Haemost. 2011;106:993–94.
- Amiral J et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia Thromb Haemost 1992;68:95–6.
- Warkentin TE et al. Treatment and prevention of heparin-induced thrombocytopenia: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133:340S–80S.
- Keeling D et al. The management of heparin-induced thrombocytopenia. Br J Haematol 2006;133:259–69.
- Warkentin TE et al. Impact of the patient population on the risk for heparin-induced thrombocytopenia. Blood 2000; 96:1703–38.
- Warkentin TE et al. Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: How much class do we need? J Lab Clin Med 2005;146:341–6.
- Whitlatch NL et al. Validation of the high-dose heparin confirmatory step for the diagnosis of heparin-induced thrombocytopenia. Blood. 2010;116:1761–6.
- Davidson SJ, Ortel TL, Smith LJ. Performance of a new, rapid, automated immunoassay for the detection of anti-platelet factor 4/heparin complex antibodies. Blood Coagul Fibrinolysis. 2011; 22:340–4
- Sachs UJ et al. Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT). Thromb Haemost 2011;106:1197–202.