A Novel, Colorimetric Platelet Activation Assay for Diagnosis of Heparin Induced Thrombocytopenia
Authors: Prechel M. Margaret , Escalante Vicki , Drenth Amanda F. , Walenga Jeanine M. 
Published: October 2019

Abstract
Abstract 5127
Washed platelet assays are used in the laboratory diagnosis of Heparin Induced Thrombocytopenia (HIT). Commonly, platelet activation is assessed by qualitative observation of platelet aggregation over time, or by measurement of radiolabeled granule release. This study describes a different washed platelet assay endpoint, using metabolic dye reduction to distinguish platelets that have been activated from those remaining at rest. The indicator reagent (CellTiter 96® AQueous One Solution, Promega Corp., Madison, WI) contains an electron coupling reagent and a tetrazolium compound which is reduced by viable cells into a colored formazan product. Washed platelets metabolize the indicator dye in a concentration- and time-dependent manner. The novel assay we have developed for application to HIT diagnosis is based on the observation that subsequent to activation by HIT-antibody immune-complexes, platelets lose the ability to reduce the dye. Wells containing platelets incubated with HIT antibodies remain light in color in the presence of 0.1 U/ml unfractionated heparin (UFH). In the presence of HIT antibody and 100 U/ml UFH, or when incubated with HIT-negative sera, platelets reduce the reagent to its dark purple product. The performance of this colorimetric assay was compared to the Serotonin Release Assay (SRA) in 17 separate assays, using a total of 145 serum specimens. For SRA, sera were incubated with 14C-serotonin labeled platelets and 0.1 or 100 U/ml UFH. Simultaneously, assays using the same specimens and conditions were conducted with unlabeled platelets prepared from the same donor. Following the incubation period, supernatants of labeled platelets were counted for radioactivity, and unlabeled platelets were exposed to reagent dye for 30–90 minutes at 37°C, then evaluated spectrophotometrically at 490 nm. Specimens positive by SRA cause platelet activation in the presence of 0.1 U/ml UFH, but not in the presence of 100 U/ml UFH. Platelet activation in the SRA has been defined historically as ≥20% serotonin release; however, higher cut-off values are often more specific for clinically relevant antibodies. Metabolic activity of post-incubation platelets is presented as the ratio of the optical density (OD) measured in wells with 0.1 U/ml UFH to the OD of wells with 100 U/ml UFH. HIT-positive specimens are detected by a low OD ratio; negative specimens result in a ratio approaching unity. Table 1 compares results of the SRA and the colorimetric assay. The mean metabolic activity ratio is >0.800 for SRA negative specimens, and decreases progressively as percent serotonin release increases. Using cut-off values of >20% serotonin release and OD ratio of <0.5, the colorimetric assay results were concordant with SRA results for 139 of145 (96%) specimens. At cut-off values of >80% serotonin release and metabolic activity ratio of <0.250, results agreed in 144 of 145 (99%) specimens. These results demonstrate a novel application of a colorimetric, metabolic indicator more commonly used as a cell viability marker. Here the dye is utilized subsequent to platelet incubation with test sera, to detect platelets that have been activated by HIT-antibody immune-complexes. Like the SRA, the assay described here is a high complexity procedure requiring washed platelets from known reactive donors. A highly significant advantage is that this assay does not require the use of radioactivity. While the results of this colorimetric assay can be appreciated by visual inspection, calculation of the OD ratio between 0.1 and 100 U/ml UFH wells allows for a less subjective, quantitative evaluation of the results. Further studies will be undertaken to establish the most relevant OD ratio cut-offs, and to determine how well this improved assay will perform in clinical practice.Table 1.Washed Platelet Activation Assay: Comparison of SRA response to Metabolic Activity RatioSerotonin Release with 0.1 U/ml UFH1nOD 490 with 0.1 U/ml UFH 2OD 490 with 100 U/mlRangeDye Incubation30 min60 min90 min0–20%1330.824 ± 0.010.808 ± 0.010.810 ± 0.0121–50%30.532 ± 0.070.691 ± 0.040.659 ± 0.1051–80%40.464 ± 0.050.428 ± 0.070.424 ± 0.1281–100%50.152 ± 0.030.105 ± 0.020.078 ± 0.031< 20% release with 100 U/ml UFH2Optical density of specimen and reagent without platelets subtracted as background

Disclosures:
No relevant conflicts of interest to declare.