Elevated thrombopoietin and platelet indices confirm active thrombopoiesis but fail to predict clinical severity of puumala hantavirus infection

Laine, Outi; Joutsi-Korhonen, Lotta; Lassila, Riitta; Huhtala, Heini; Vaheri, Antti; Mäkelä, Satu; Mustonen, Jukka

For TPO, blood samples were collected and centrifuged at 1700g for 15 minutes, and the serum was frozen at À70 0 C. Human Thrombopoietin Immunoassay Quantikine ELISA was applied according the manufacturer's instructions (R&D Systems, Minneapolis, MN). In healthy volunteers (n = 38), the mean level of serum TPO is 74.2 pg/mL (ranging from nondetectable to 228 pg/mL), according to the manufacturer. To measure procoagulant activity of MPs in plasma, citrate-anticoagulated (109 nmol/L sodium citrate) blood samples were centrifuged at 2000g for 15 minutes. Plasma was then separated, further centrifuged at 13,000g for 2 minutes, and frozen at À70 0 C. Blood sampling and processing of plasma were in accordance with the instructions of ZYMYPHEN MP-Activity ELISA (ANIARA, HYPHEN BioMed, West Chester, OH) that was applied for analyses. The MP-Activity test kit is a functional assay measuring procoagulant activity of MPs derived from various cellular sources. [12] In the assay, circulating MPs in the plasma sample bind to Annexin V on the microplate surface and expose their phospholipid surface, which allows prothrombin activation by factor Xa and Va complex in the presence of calcium. The exposed phospholipid concentration reflects the amount of thrombin generation, which is measured using specific thrombin substrate. According the manufacturer, concentration of MPs in normal plasma is usually 5 nmol/L (expressed as phophatidylserine equivalent).