A-166 Performance verification of a polyclonal anti-PF4 immunoassay in evaluation of Heparin Induced Thrombocytopenia (HIT)
Authors: Arnouk J , Colby S , Williams R , Olinski J , Barbhuiya M 
Published: October 2024

Abstract

Background
Heparin-induced thrombocytopenia (HIT) is a severe complication of exposure to heparin that occurs in a small percentage of patients in anticoagulation therapy. HIT could happen regardless of the dose, exposure, schedule, or route of administration. HIT results from an autoantibody directed against endogenous platelet factor 4 (PF4) that forms complex with heparin. This antibody activates platelets and can cause life threatening arterial and venous thrombosis. Untreated HIT has a mortality rate as high as 20%. However, with appropriate screening, diagnosis and early intervention, mortality rates have been reported to be below 2%. The laboratory evidence in evaluating patients for HIT is crucial for early intervention. There are several assays available to diagnose Heparin Induced Thrombocytopenia (i.e., immunoassay and/or functional assay for HIT antibodies; serotonin release assays). The purpose of this study is to establish the measurement criteria and verify performance characteristics of a polyclonal anti-PF4 immunoassay. The test of interest (anti-PF4 immunoassay) is intended for the qualitative detection of the anti-heparin-platelet factor 4 antibodies in plasma or serum by ELISA.

Methods
The ASSERACHROM Assay (Diagnostica Stago, Inc. USA) was implemented in the automated Dynex DS2 ELISA Processing System (Dynex Technologies Inc USA). Two Dynex DS2 instrument comparisons along with precision, accuracy and measurement range for qualitative positive and negative results were studied. The reference method comparisons with serotonin release assay and other monoclonal anti-PF4 immunoassays were performed. To evaluate performance of the assay, we conducted precision, accuracy, and qualitative method comparison. We compared the assay of interest with two reference lab immunoassays and the gold standard reference method (serotonin release assay). The statistical analysis was carried out in EP Evaluator and GraphPad Prism.

Results
The negative control precision demonstrated CV of 9.2% (N=20, mean= 0.11, SD= 0.01). The analyte measurement is expressed as optical density (OD). The positive control precision characteristics showed CV of 8.1% (N=20, mean= 1.62, SD= 0.13). The threshold was defined as reference reagent absorbance (R6OD) times the variance factor divided by 100 ((R6OD* variance factor)/100= threshold (X)). This is used for positive (&gt;the X) and negative (&lt; the X) absorbance cut off for patient samples. The R6 reference material precision study resulted in a CV of 7.95% (N=20, mean= 2.1, SD= 0.17). The R6 variance factor per precision statistics at 3SD level was 23.85%. This is comparable to peer group variance provided in the assay package insert (23.5%). The polyclonal anti-PF4 immunoassay versus a reference monoclonal method (N=40) demonstrated agreement of 90.9% with positive agreement of 100% and negative agreement of 89.5%. When compared with gold standard serotonin release assay (N=40), the assay demonstrated agreement of 75% with positive agreement of 90% and negative agreement of 60%. The two instruments demonstrated agreement of 97.5% with positive agreement of 100% and negative agreement of 93.3% (Cohen's Kappa 94.6%; with Kappa &gt; 75% indicates “high” agreement).

Conclusions
This anti-PF4 immunoassay is precise, accurate and comparable with three different reference methods. The test is best utilized when 4T score based clinical algorithm is used for evaluation of HIT.