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Heparin-induced thrombocytopenia (HIT) is a ‘clinicopathologic’ syndrome (i.e. diagnosis depends on both clinical and pathologic criteria being present) [1]. The ‘clinical’ criteria include the presence of thrombocytopenia and/or thrombosis bearing a temporal relationship to an immunizing exposure to heparin. The ‘pathological’ criterion is the detectability of ‘HIT antibodies’ in acute patient serum or plasma. HIT is caused by heparin-dependent platelet-activating antibodies that in almost all patients recognize complexes of platelet factor 4 (PF4) bound to heparin [2]. Sensitivity of solid-phase enzyme-immunoassays (EIAs) for anti-PF4/heparin antibodies is very high (approximately 99%), due to certain unique properties of HIT antigens; they are expressed on large, stable multimolecular complexes comprised of PF4 and polyanions [3]. This differs from other drug-induced, immune-mediated thrombocytopenic disorders, where laboratory tests for drug-dependent antibodies often lack high diagnostic sensitivity. The antigens in these other disorders are labile complexes of drug (or drug metabolites) and platelet glycoproteins [4].

The high diagnostic sensitivity of the PF4/heparin EIAs is accompanied by a parallel problem in diagnostic specificity for HIT. This arises because EIAs also have high sensitivity for detecting clinically insignificant anti-PF4/heparin antibodies, which can be present coincidentally in patients with thrombocytopenia caused by non-HIT factors. This leads to the potential for HIT ‘over-diagnosis,’ especially in critically-ill patients. Systematic serosurveillance studies show that only a minority (2–15%) of heparin-treated patients who form anti-PF4/heparin antibodies develop clinically evident HIT [5,6]. Even among those who form platelet-activating antibodies, no more than half develop HIT. This Subcommittee statement provides a framework on when to perform and how to interpret laboratory tests for HIT diagnosis.

Patients should not be routinely screened for anti-PF4/heparin antibodies (other than for research studies). Patients should only undergo testing if clinical features reasonably suggest a diagnosis of HIT. Scoring systems can be helpful to estimate the pretest probability of HIT [7–9]. HIT antibodies are transient, however [10], and thus testing should be performed using acute serum or plasma.

Laboratory diagnosis of HIT differs fundamentally from other antibody-mediated cytopenias, such as autoimmune thrombocytopenia or hemolysis, because free HIT antibodies are readily detectable in patient serum/plasma even during the earliest phase of the platelet count decline indicating HIT [11]. Therefore, a negative result of an HIT antibody test performed because of thrombocytopenia or thrombosis generally rules out HIT. Routine repeat testing a few days later is not indicated (unless a new platelet count decline or thrombosis occurs) because such testing risks detecting clinically irrelevant antibodies (as demonstrated in serosurveillance studies). Over-diagnosis of HIT is potentially dangerous because it may lead to withholding of certain diagnostic and therapeutic interventions during the acute event and in future admissions, and because it usually prompts treatment with alternative anticoagulants, which may increase bleeding risk (approximately 1% risk of major bleeding per treatment day for direct thrombin inhibitors) [12].

Diagnostic accuracy for HIT is optimized by combining the anti-PF4/heparin EIA with a functional (platelet activation) test. The major advantage of the PF4-dependent EIAs is that due to their high sensitivity, a negative result essentially rules out HIT (high negative predictive value). Platelet activation assays, particularly when performed using ‘washed’ platelets (e.g. serotonin-release assay [SRA] and heparin-induced platelet activation [HIPA] test), are much more specific for HIT than are EIAs [2,5,6,11]. Among patients who are investigated for clinically suspected HIT, only about 50% of those who test EIA-positive also test positive in a washed platelet activation assay [2,13] (and only approximately 15% of critically ill patients who test EIA-positive [14]). Functional assays based on platelet-rich plasma or whole blood are less sensitive than washed platelet assays, with potential for false-negative results [15]. Whereas this Working Group recommends the approach of dual EIA-platelet activation testing (at least for reference laboratories), it is recognized that in many centers the first-line test is the immunoassay, in which case the functional assay could be restricted to patients testing EIA-positive (and especially when the EIA result is not strongly positive, as discussed subsequently).

There are several maneuvers to improve diagnostic specificity of EIAs. Patients should be tested for IgG class antibodies, as only this isotype class activates platelets [2,6,16,17] (rare exceptions may exist with pathogenic IgA and/or IgM antibodies, but this is controversial). EIA results should be expressed quantitatively in graded optical density (OD) ranges, because the strength of a positive EIA test result (expressed in OD units) correlates strongly with the presence of platelet-activating antibodies [13,17] and the risk of thrombosis [18] (even so, approximately 5–15% of sera yielding a weak-positive result, i.e. OD < 1.0 in the EIA [the respective thresholds may differ among EIAs], are platelet-activating [13,19]). Repeating the EIA in the presence of high heparin concentrations (100 IU mL−1) increases specificity somewhat [20], especially at OD levels < 1.0 (however, caution is advised in excluding HIT by this maneuver when the OD level is higher, because strong platelet-activating antibodies may not be inhibited by high heparin [21]). Commercial PF4-dependent EIAs differ with respect to their two key components, PF4 (recombinant vs. platelet-derived, either purified or from platelet lysate) and polyanion (heparin, biotinylated heparin or polyvinylsulfonate). While this seems not to influence detectability of anti-PF4/heparin antibodies greatly, the different assay designs may affect the interpretation of OD levels and inhibition by high heparin.

Rapid screening for HIT antibodies can be performed using PF4-dependent particle-based immunoassays. One such assay, the particle gel immunoassay, has the advantage of higher diagnostic specificity than the EIAs (although lower than the platelet activation assays), but the disadvantage of giving false-negative results in approximately 5% of HIT cases [22]. The clinical usefulness of this assay is enhanced when combined with use of a clinical scoring system [23]. Another rapid particle-based immunoassay, the particle immunofiltration assay, showed poor predictivity for HIT antibodies in two laboratories [24], and its use cannot be recommended.

With respect to platelet activation assays, improvements in test specificity can result from increasing the threshold for indicating presence of clinically significant antibodies, for example using a serotonin-release threshold of > 50% (instead of > 20%) [13], or a shorter time to platelet aggregation (lag time < 30 min instead of < 45 min) in the HIPA test. The functional assays require strict quality controls; most important is the inclusion of a weak-positive HIT serum control (to ensure adequate platelet reactivity in maintaining high test sensitivity), the use of high heparin inhibition, and an appropriate negative control [25]. Approximately 2–3% of sera consistently cause strong platelet activation at both low and high heparin concentrations; here the functional assay is non-informative (‘indeterminate’ result) [26].

High diagnostic specificity is especially important during evaluation of efficacy and safety of novel therapies for HIT. It is a recommendation of the Platelet Immunology SSC Working Group on HIT that a diagnosis of HIT requires a clinical scenario in which HIT is judged to be the most plausible diagnosis, generally meeting at least an intermediate probability for HIT (judged by a clinical scoring system), plus either a strong-positive EIA-IgG or, preferably, a positive test for platelet-activating antibodies. Frozen HIT sera remain stable for many years, making such testing feasible even in retrospect.

Disclosure of Conflict of Interests

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T.E. Warkentin has provided consulting services to and/or has received research funding from Gen-Probe GTI Diagnostics and DiaMed. A. Greinacher has provided consulting services to and/or has received research funding from IL Laboratories and DiaMed.

References

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