title: A colorimetric, metabolic dye reduction assay detects highly activated platelets: application in the diagnosis of heparin-induced thrombocytopenia.
abstract: Heparin-induced thrombocytopenia (HIT) is caused by antibodies, elicited in response to heparin anticoagulant therapy, which can cause extreme platelet activation and result in a highly procoagulant state. Most diagnostic tests for HIT antibodies measure in vitro platelet activation by detecting aggregation or granule release responses. This study demonstrates that the high level of activation by HIT antibodies leads to a rapid breakdown of platelet metabolic activity. Resting or mildly activated platelets metabolize tetrazolium-based indicator dye to a dark colored product, in proportion to cell concentration and dye incubation time. Highly activated, procoagulant platelets resulting from incubation with HIT antibodies fail to metabolize dye and remain light in color. The loss of ability to metabolically reduce indicator dye provides a colorimetric endpoint that can be used in an in vitro washed platelet activation assay to detect HIT antibodies. In a prospective evaluation, 145 diagnostic specimens were tested concurrently by both the colorimetric, dye reduction assay and the clinical laboratory standard, radiolabeled-serotonin release assay ((14)C-SRA). Results were in agreement for 96-100% of cases, depending on the chosen stringency of assay cut-off values. This study demonstrates that the metabolic dye reduction assay is comparable to the (14)C-SRA for HIT diagnosis. In addition, this novel assay may have even wider applicability, facilitating studies on the physiologic and clinical relevance of highly activated platelet populations.
authors: Department of Pathology, Loyola University Medical Center, 2160 SouthFirst Avenue, Maywood, IL 60153, USA. mpreche@lumc.edu
authors: M Margaret
authors: MM
authors: Prechel
authors: Vicki
authors: V
authors: Escalante
authors: Amanda F
authors: AF
authors: Drenth
authors: Jeanine M
authors: JM
authors: Walenga
mesh_terms: D000925:Anticoagulants / Q000009:adverse effects*; D001792:Blood Platelets / Q000378:metabolism*; D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry; D005260:Female; D005562:Formazans / Q000737:chemistry; D006493:Heparin / Q000009:adverse effects*; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction; D015539:Platelet Activation; D010979:Platelet Function Tests / Q000379:methods; D013921:Thrombocytopenia / Q000097:blood / Q000139:chemically induced / Q000175:diagnosis
chemical_list: D000925:Anticoagulants; D004396:Coloring Agents; D005562:Formazans; D006493:Heparin