A Radiolabeled PF-4 Staph-a Binding Assay Proposed As a Solitary Method for Confirming Heparin Induced Thrombocytopenia
Authors: Schwartz Kenneth A. , Davis John , Tokala Hemasri 
Published: October 2019

Abstract
Introduction:Heparin induced thrombocytopenia (HIT) is present in over 30% of normal subjects receiving heparin and the incidence of HIT may even be higher in patients with occlusive atherosclerotic disease. Confirmatory laboratory assays can broadly be classified as functional which have high specificity and rely on activation of platelets by the platelet factor 4 (FP4)-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, after the episode of decreased platelets is resolved, an increase in the patient’s platelet count 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. Our working hypothesis is that since this assay has both immune and binding components it would have better specificity and sensitivity than the currently used assays and as a single assay be more cost effective. 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, to record the results of the hospitals’ assays for HIT and to ascertain what other causes of thrombocytopenia might be possible.
Results: To date we have evaluated 20 samples. True positives were observed in 3 hospital patients and 4 known positives that were a gift from the Blood Center of S.E. Wisconsin. 10 true negatives included 5 from hospital patients and 5 from thrombocytopenic patients who were not treated with heparin. 3 samples were negative in our assay, had a 4 ”T” score of 6 and 5 and NA and had increased platelet counts after heparin was stopped and were judged as false negatives. However, these samples had high relative binding to the Staph-A-Sepharose in the sample without the addition of heparin suggesting that either the sample already had enough heparin from the patient’s treatment to form immune complexes or as has been recently reported another negatively charged molecule formed a complex with PF-4.(Warkentin Blood;2014;123:3651) Concordance between the radiolabeled PF-4 assay and the commercial PF-4 hospital assay was observed in 10 of 11 patients.
Conclusions:To date when judged using clinical criteria, the radiolabeled PF-4 assay correctly distinguished true positives and true negatives in 17 of the 20 samples. This is an ongoing study with the goal of studying another 30 patients before the ASH December meeting. The addition of clinical criteria including the 4 “T” score and the increase in platelet count after stopping heparin to a review of the patient’s acute care hospital record is helpful in evaluating the reliability of the radiolabeled PF-4 assay as a predictor of HIT.

Disclosures
No relevant conflicts of interest to declare.