Clinical Validation of a Radiolabeled PF-4 Staph-a Binding Assay for Confirming Heparin Induced Thrombocytopenia

Introduction:Confirmatory laboratory assays for heparin induced thrombocytopenia (HIT) can broadly be classified as functional which have high specificity and rely on activation of platelets by the platelet factor 4 (PF4)-heparin-IgG immune complex or as immune based assays that are relatively more sensitive. At the time when the clinician is evaluating heparin as a cause of thrombocytopenia the 4 "T" criteria are helpful. However, an increase in the patient's platelet count after the heparin has been stopped is critical for confirmation of the diagnosis.

Methods: We developed a variation of a previously described technique (Newman Thromb Haemost 1998;80:292) and used clinical criteria as the standard for comparison to evaluate the assay. Radiolabeled 125-I-PF4 is incorporated into the immune complex of PF-4-heparin-immunoglobulin and the amount of radiolabeled immune complex is measured after binding to staphylococcal A protein sepharose (Staph-A). The hospitalized subjects medical record was reviewed to: measure a 4 "T" score, to determine if the patient's platelet count increased after heparin was stopped and to exclude other plausible causes of thrombocytopenia.

Aim: The assay relies on the binding of the heparin immune complexes to Staph A. Staph A preferentially binds to larger as compared to smaller immune complexes and the larger complexes produce a greater degree of F(c) mediated platelet activation when compared with the smaller complexes. This suggests the hypothesis that a Staph A based assay will have have better specificity and sensitivity than the currently used methodologies.

Results: 28 patient samples were evaluated. True positives were observed in 4 hospital patients and 4 known positives. 19 were true negatives and included 7 from hospital patients and 12 from thrombocytopenic patients who were not treated with heparin. 1 sample was negative in our assay, and was judged as false negative. Concordance between the radiolabeled PF-4 assay and the commercial PF-4 assay was observed in all the 28 patients.

Conclusions: To date when judged using clinical criteria, the radiolabeled PF-4 assay correctly distinguished true positives and true negatives in 27 of the 28 samples.