Regular ArticleEvaluation of automated immunoassays in the diagnosis of heparin induced thrombocytopenia
Introduction
Heparin-induced thrombocytopenia (HIT) is a prothrombotic, immune-mediated adverse reaction that occurs after exposure to unfractionated heparin (UFH), low molecular heparin (LMWH), or other polyanions such as hypersulfated chondroitin sulphate. HIT is usually caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin-complexes [1], [2]. Typical clinical manifestations of HIT are a fall in platelet count by more than 50%, beginning between day 4 to 10 of heparin therapy, often complicated by new thrombotic complications [3], [4], [5]. However, the clinical diagnosis of HIT is often difficult, especially in patient populations with a high prevalence of thrombocytopenia such as intensive care patients [6].Thus, the clinical diagnosis must be corroborated by in vitro demonstration of heparin dependent antibodies, e.g. platelet activating and/or anti-PF4/heparin IgG antibodies [3], [7], [8].
Two different classes of assays are available: enzyme immunoassays (EIAs), which detect binding of antibodies to immobilized PF4/heparin complexes and functional assays, which investigate the capability of these antibodies to activate platelets in the presence of heparin. Although functional assays such as the serotonin release assay (SRA) [9] or the heparin-induced platelet activation assay (HIPA) [10], are considered to be the gold standard in the laboratory diagnosis of HIT, they are technically challenging and restricted to specialized laboratories [11]. In contrast, immunoassays are easy to perform and widely available. Because of their excellent negative predictive values (NPV), immunoassays are helpful in excluding HIT. However, these assays usually do not differentiate between pathogenic, i.e. platelet activating, antibodies and clinically irrelevant antibodies and HIT is grossly overdiagnosed when only the result of the antigen assay is considered [11]. Immunoassays detecting IgG antibodies only have improved operating characteristics by increased diagnostic specificity for pathogenic antibodies [12], [13], [14], however, still only ~ 50% of anti-PF4/heparin IgG antibodies are platelet activating. An additional approach to increase specificity of PF4/heparin antibody assays is to take the magnitude of the test reactivity (usually given in optical density [OD] values) into account, as it correlates with the likelihood of HIT determined by a scoring system and with the capability of the antibodies to activate platelets [12], [14]. Beside their low positive predictive values for HIT, another disadvantage of the currently available immunoassays results from the preferred testing in batches rather than single patient samples for cost considerations and from the lack of standardization.
Three automated PF4/heparin antigen assays, one based on the agglutination of latex particles (HemosIL® HIT-Ab(PF4-H)) and the two others with chemiluminescent detection (HemosIL® AcuStar HIT-Ab(PF4-H) and HemosIL® AcuStar HIT-IgG(PF4-H), respectively), have recently been introduced. In the latex agglutination assay, agglutination of PF4/polyvinylsulfonate coated beads by a monoclonal anti-PF4/heparin-antibody is inhibited in the presence of human anti-PF4/heparin antibodies [15]. This assay can only be performed with plasma (the only sample matrix suitable for the ACL TOP® Family System) and it cannot differentiate between IgG, IgM, or IgA antibodies. The chemiluminescence assays are based on binding of anti-PF4/heparin antibodies to PF4/polyvinylsulfonate. They can be performed with serum or plasma and can differentiate between different immunoglobulin classes (IgG only or total antibody). They show a wide range of reactivity and might thus provide additional information compared to the EIAs [16]. These automated assays might allow greater standardization and better comparability of results obtained in different laboratories. Furthermore, they allow single sample testing and provide a rapid turn around time of results of approximately 13 minutes for the latex immunoassay and about 30 minutes on the chemiluminescent system.
In this study we assessed these three assays in comparison with an in-house PF4/heparin EIA and the HIPA test.
Section snippets
Study design
Blood samples from 491 consecutive surgical and medical patients who received UFH or LMWH and in whom HIT was suspected were evaluated in this study. 94 suspected HIT plasma samples were first referred to the laboratory of the University Hospital of Florence, Italy and 34 suspected HIT plasma samples to the laboratory of the Royal Brompton Hospital, London, United Kingdom. These samples were tested on the day of arrival using the latex agglutination test (HemosIL-Ab). Then the samples were
Latex agglutination test
In total 119 citrated plasma samples from individual patients were tested in the agglutination test (HemosIL HIT-Ab) that detects IgG, IgM, and IgA antibodies against PF4/hep and nine samples were excluded due to insufficient sample volume. 44 (37%) samples tested positive, among which 20 (17%) were also positive in the HIPA.
Chemiluminescence-based assays
In total 491 samples of consecutive patients were enrolled, 43 patients were excluded because of insufficient sample volume (n = 24 serum samples, n = 9 plasma samples) or
Discussion
This study shows that the new, highly standardized automated antigen assays for PF4/heparin antibodies have the highest positive predictive values for platelet activating antibodies of all antigen assays so far reported, ranging from 41.8% for the HemosIL AcuStar-Ab assay to 78.1% for the HemosIL AcuStar-IgG test. Especially, a strongly positive result in these assays with reactivity of more than 4 U/mL is associated with a likelihood of > 85% that the sample contains platelet activating and
Conflict of interest statement
Instrumentation Laboratory provided test reagents and ACL AcuStar™.
Acknowledgements
This study has been supported by: Instrumentation Laboratory (test reagents and ACL AcuStar).
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2020, BloodCitation Excerpt :Evaluation of the 3 IAs revealed that the sensitivities of the recommended cutoffs differ significantly: 100% for ID-PaGIA-H/PF4, 96.2% for Zymutest-HIA-IgG, and 80.8% for the HemosIL-AcuStar-HIT-IgG (supplemental Table 1). With regard to this last IA, we confirm38,46 that the official cutoff value set at ≥1.0 U/mL is too high, resulting in a not inconsequential number of false-negative results (13 of 101 HIPA-positive samples) (supplemental Table 4). Of note, Althaus et al46 previously observed a sensitivity of 96.2% for the official cutoff of the HemosIL-AcuStar-HIT-IgG, identifying an ideal cutoff according to ROC analysis of 0.57 U/mL, which is similar to our data.