The ultrasound-guided LV puncture method of blood drawing minimizes in vitro release of TGF-β1, PF4, and TSP-1 from platelet α-granules into plasma. (A) Blood samples were drawn into sodium citrate anticoagulant by either the RB capillary technique in the absence (RB; n = 25) or presence of 1μM PGE₁ (RB + PGE₁; n = 10), or by the ultrasound-guided LV method (n = 15). Plasma samples were prepared by centrifuging blood at 3000g for 15 minutes at RT. Total TGF-β1 was measured by ELISA. (B) TSP-1 and (C) PF4 antigen levels were estimated by measuring immunoreactive band intensities on immunoblots. Each dot represents an individual mouse sample, and each color represents data obtained from the same mouse. P values reflect RB values compared with LV values. (D) Representative immunoblots with antibodies to TSP-1 and PF4 of plasma prepared by the RB method from 6 mice and by the LV methods from 5 different mice. Bottom panel: A nonspecific band, indicating that similar amounts of protein were loaded in each lane. (E) Total TGF-β1 was measured in plasma prepared from blood collected first by the LV method and subsequently by the RB method (left panel) or from blood collected in the opposite sequence (right panel).