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Laboratory Evaluation of Heparin-Induced Thrombocytopenia
Heparin-induced thrombocytopenia (HIT) is a clinicopathologic syndrome characterized by a constellation of clinical findings and identification of antiplatelet factor 4 (PF4)/heparin antibodies. Classically in HIT, progressive thrombocytopenia is observed, reaching a nadir within 5–10 days following the immunizing heparin exposure. In addition, because the mechanism by which thrombocytopenia ensues involves platelet activation, thrombosis occurs in a significant proportion of cases. Thrombosis in HIT can be severe and life-threatening. As a result, a decrease in platelet count by 50% after heparin administration is sufficient evidence to replace heparin with an alternative anticoagulant pending laboratory evaluation of HIT unless a rapid assay is available. As heparin is usually a safe and often preferred drug for inpatient thromboprophylaxis, cardiac surgery, and other interventional procedures, laboratory assessment of HIT is valuable in distinguishing true cases of HIT from thrombocytopenia of other etiologies.
Understanding of the molecular mechanisms of HIT has led to a model in which antibodies are formed against a complex between PF4 and heparin. These pathogenic anti-PF4/heparin antibodies provoke platelet activation by interaction with the platelet FcγIIa receptor, resulting in platelet aggregation, thromboxane generation, granule release, formation of platelet-derived microparticles, and overall stimulation of a procoagulant response. Other mechanisms potentially contributing to hypercoagulability include activation of endothelial cells and monocytes with resultant exposure of tissue factor and neutralization of the anticoagulant effect of heparin by released PF4, which further propagates thrombogenesis.
Functional assays such as the serotonin release assay (SRA) or heparin-induced platelet activation assay (HIPA) are considered the gold standard for laboratory diagnosis of HIT because of high sensitivity and specificity. However, these assays are difficult to perform, requiring, in some cases, specialized equipment and use of radioactivity, as well as well-characterized platelet donors and experienced operators. Thus, SRA and HIPA are not feasible as general screening tests for anti-PF4/heparin antibodies. Accordingly, a number of different methodologies have been developed to allow routine screening for anti-PF4/heparin antibodies. These assays have high sensitivity, but in some cases can result in a substantial rate of false positives because of lower specificity. Currently, the best approach is considered to be identification of anti-PF4/heparin antibodies through a combination of an antigen assay and functional platelet activation assay, if required, interpreted in conjunction with the clinical pretest probability (e.g., 4Ts score).
Antigen Assays
In general, most clinical laboratories utilize commercially available immunoassays for detection of anti-PF4/heparin antibodies. These assays detect anti-PF4/heparin antibodies using PF4 as a substrate. To simulate in vivo epitopes, PF4 is complexed with either heparin or other polyanion molecules that mimic heparin.
Solid-Phase Enzyme Immunoassays
In solid-phase enzyme immunoassays (EIA), PF4/polyanion complexes are bound to microtiter plates, and after incubation with patient sample and addition of an enzyme-linked antihuman globulin reagent, a colorimetric response can be quantified using an automated microplate reader. The resultant optical density (OD) correlates with a qualitative (positive or negative) result depending on a prevalidated cutoff. However, subtle differences in commercially available kits exist, regarding the source of PF4 (recombinant vs. platelet-derived) and the use of heparin or other polyanions such as polyvinyl sulfonate (PVS). One commercially available EIA incorporates the addition of interleukin-8 (IL-8) and neutrophil-activating peptide 2 (NAP-2), two chemokines that have been implicated in cases of HIT. Despite these differences, solid-phase EIAs have sensitivities approaching 94%–100%; however, there is a significant variability in clinical specificity. Two recent systematic reviews and meta-analyses have examined the diagnostic value of anti-PF4/heparin immunoassays.
In the past, immunoassays incorporated combinations of antihuman IgG, IgA, and IgM, and thus detected anti-PF4/heparin antibodies of IgG, IgM, and IgA isotypes. However, it is now understood that the pathogenesis of HIT relies on antibody binding to the platelet FcγIIa receptor, which is IgG dependent. Although isolated cases of HIT associated with IgA and IgM anti-PF4/heparin have been described, these antibody isotypes are not typically considered pathogenic and acceptance of positive results without confirmation by a functional assay may be partly responsible for the overdiagnosis of HIT. As a result, use of IgG-specific assays have increased the clinical specificity of antigen assays. Guidelines from the International Society on Thrombosis and Haemostasis and British Committee for Standards in Haematology/British Society for Haematology advocate for the use of IgG-specific EIAs. In addition, to increase specificity EIAs can also be performed following incubation with high concentrations of heparin (10–100 IU/mL). Reactivity of anti-PF4/heparin antibodies is inhibited at high heparin concentrations.
As an adjunct to guide further testing, a recent publication correlated OD measurements of solid-phase EIAs to SRA. In this study, the probability of pathogenic antibodies as defined by strong positivity (>50% release) in the SRA increased in relation to the magnitude of OD measurements of the EIA. It was determined that most cases of HIT were associated with an EIA OD 1.40 units, whereas weak positive results (0.40–1.00 OD units) typically excluded the diagnosis of HIT. Because of lower EIA OD cutoffs supplied by manufacturers, the author recommends reporting the qualitative and quantitative result.
Fluid-Phase Enzyme Immunoassays
As a result of the inherent problem of protein denaturation with solid-phase EIAs, a fluid-phase EIA in which anti-PF4/heparin IgG bind PF4/heparin antigens followed by capture with beads was developed. Although not commonly used clinically, this assay may have greater sensitivity than the aforementioned solid phase EIAs. In addition, this method avoids nonspecific binding by minimizing exposure of cryptic antigens affiliated with denatured PF4.
Rapid Immunoassays
For rapid detection of PF4/heparin antibodies, additional assays such as the particle gel immunoassay (PaGIA), particle immunofiltration assay (PIFA), a latex agglutination assay, and chemiluminescent immunoassays have been added to the armamentarium of HIT testing. With analytical turnaround times less than 30 minutes and on demand availability, these assays allow clinicians to make an informed decision before switching to alternative anticoagulation, thereby improving outcomes while decreasing costs.
Particle Gel Immunoassay
In the PaGIA, PF4/heparin complexes are bound to high-density polystyrene beads. If present, anti-PF4/heparin antibodies bind to the beads and are detected through agglutination that occurs as a result of the addition of a secondary antihuman globulin reagent. Similar to gel testing widely used in blood banks, agglutinated beads fail to migrate through the gel, producing a visible band that indicates a positive result. Because of limited data, the sensitivity and specificity of this assay is currently uncertain; however, it is postulated to be intermediate between solid-phase EIAs and functional platelet assays.
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