•A novel antithrombotic formulation blood collection tube allows simultaneous analysis of thirty-two blood analytes.•The hematological, biochemical and d-dimer measurands can be measured in a single blood collection tube.•A new formulation has a potential to allow measurements of glucose and homocysteine due to stabilized blood cell metabolism. The objective of our study was to evaluate a single blood collection tube with a novel antithrombotic formulation to measure both hematological, biochemical, and d-dimer analytes. Paired samples of gold standard blood tubes (EDTA, lithium heparin, sodium citrate) and a new antithrombotic formulation blood tube were collected from 187 patients. The new antithrombotic tube is a lithium heparin tube preloaded with a liquid form of prostacyclin analog. The novel tube was tested on seventeen hematological parameters and smears against EDTA, on fourteen biochemical parameters against lithium heparin and on d-dimer against sodium citrate. All correlation coefficients were close to 0.99. The Bland-Altman analyses presented a satisfactory correspondence for all analytes. All the hematological examinations demonstrated comparable results between EDTA and the novel formulation, except for platelet counts analyzed by impedance method, but not by fluorescence. We detected lower mean platelet volume with/without outliers (5.06%)/(5.13%) in the novel formulation and increased mean corpuscular hemoglobin concentration (2.55%). All the biochemistry analytes demonstrated comparable results between lithium heparin and the novel tube. d-dimer showed comparable results between citrated blood and the novel formulation after dilution correction. We describe a novel antithrombotic formulation tube with the potential to be introduced into clinical laboratories for simultaneous analysis of thirty-two blood analytes.