Better Accuracy for the Laboratory Detection of HIT Antibodies Using IgG-specific ELISA Assays

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin therapy — a common anticoagulant treatment that induces prothrombotic complications. The prothrombotic mechanism arises from the formation of platelet-activating IgG-specific antibodies to the heparin-platelet factor 4 (PF4) complex. This complex of heparin-PF4 (H-PF4) and IgG specific antibodies binds to the platelet Fc receptor causing activation and release of granules from the platelet. The platelet count will decrease as the platelets are activated and the patient is at risk for developing thrombotic complications. The pretest probability of HIT versus other causes of thrombocytopenia is initially evaluated by assessing the clinical picture and the timing and degree of platelet count decrease, embodied in the 4T’s score. The widely used laboratory assessment for HIT antibodies is an enzyme-linked immunosorbent assay (ELISA) that detects antibodies against H-PF4. These assays are highly sensitive; however, they have only moderate specificity. The lower specificity is due to the nonpathogenic antibodies being indistinguishable from the true HIT-causing antibodies. IgG, IgA, and IgM antibodies against the H-PF4 complex can be detected, but only the IgG antibody class can bind to the Fc receptor and cause subsequent platelet activation, thrombocytopenia, and the clinical prothrombotic state.

Initially, the available commercial and laboratory-developed tests (LDT) for HIT antibodies detected all three classes of antibodies. Now, IgG-specific commercial tests and LDTs have been developed in an attempt to increase the diagnostic specificity for HIT. Both IgG-specific and polyspecific antibodies ELISA assays are available. However, there is considerable confusion and contradictory information about which test type is better for the diagnosis of clinically significant HIT. With this controversy, the International Society of Thrombosis and Hemostasis stated a preference for the IgG-specific ELISA assay, as it most closely parallels the pathogenic mechanism of HIT that is needed for binding to the FC receptor on the platelet to cause activation. After this recommendation, a meta-analysis of essentially all the studies on HIT ELISA assays (comparing both IgG-specific and poly-specific assays) demonstrated no difference in sensitivity and specificity between IgG-specific assays and polyspecific assays.¹  This meta-analysis was too broad in its comparison of studies since it used different populations and reference standards. A second and more comparable meta-analysis was performed by Dr. Holleh Husseinzadeh and colleagues with the caveat of using only studies with direct comparability of the IgG-specific and the polyspecific ELISAs.

In the more rigorous meta-analysis assessment of HIT ELISA studies, the authors pooled the data from nine published studies where both IgG-specific and poly-specific ELISA methods were directly compared. The sensitivity of the two HIT methods were essentially identical (0.97; 95% CI, 0.95-0.99), but the IgG-specific method was significantly better for specificity compared to the polyspecific method (0.87; 95% CI, 0.85-0.88 vs. 0.82; 95% CI, 0.80-0.84). The negative predictive values (NPV) for IgG-specific and poly-specific methods were both high and similar (0.99 CI=0.99-1.00). Conversely, the positive predictive value (PPV) was significantly better for the IgG-specific method compared to the poly-specific method (0.56; 95% CI, 0.52-0.61 vs. 0.32; 95% CI, 0.28-0.35). In seven of the nine studies, the results were compared with the functional conformational assay, the serotonin release assay (SRA). The IgG-specific method and the polyspecific method still had similar sensitivity when compared to the SRA, but the specificity was still better for the IgG-specific method when compared to the SRA reference standard. Of note, many clinicians assume that all SRA results are similar, but significant variation in SRA methodology was found between these reported studies.