2.6 Hematological variables
We measured the activated partial thromboplastin time (APTT), prothrombin time, fibrinogen and D-dimer on an automated coagulation analyzer (STA evolution®, Stago, Paris, France), following protocols from the manufacturer.
White blood cell count, hemoglobin and platelet count were performed on a fully automatic analyzer (Sysmex XN 550®, Le Roche, Basel, Switzerland).
TGT was analyzed using the Genesia® analyzer and the STG®-Thromboscreen kit (Diagnostica Stago, Paris, France), based on the fluorometric method described by Hemker [21]. When testing, a new calibration test, three levels of quality control (low, normal, and high TM resistance), and a reference plasma to normalize parameters are assessed. The STG®-Thromboscreen kit contains a mixture of phospholipids and an intermediate picomolar concentration of human recombinant tissue factor, with and without adding TM, as activator of the coagulation system.TM concentration is that required to obtain a 50% decrease of the endogenous thrombin potential (ETP). The final TM concentration is not disclosed by the manufacturer. TG was measured in PPP samples thawed in a 37ª water bath for 3 minutes. TGT provides information on the start time of thrombin generation (lag time, LT minutes), the ETP or total amount of thrombin generated (ETP, nM/min), the peak of thrombin concentration (Max peak, nM), the time to reach the maximum peak of thrombin (Tt Peak, minutes), the time to thrombin neutralization (start tail, ST, minutes) and the velocity of thrombin formation (Vel Index, nM/min). We defined TM resistance as an ETP reduction less than 40% after TM addition. This definition was established home-made applying TGT to COVID-19 patients with thrombotic risk (n = 120). Patients with an ETP reduction less than 40% after TM addition had the worst outcome, in terms of thrombotic development or death. All samples were processed and analyzed at the University General Hospital Dr. Balmis.
Global hemostatic analysis in whole blood samples was also measured with the Quantra® hemostasis analyzer (Qplus® and QStat®, HemoSonics, Paris, France). This technique informs about the clot time (CT, seconds), heparinase clot time (HCT, seconds), clot stiffness (CS, hPa), fibrinogen contribution to clot stiffness (FCS, hPa), platelet contribution to clot stiffness (PCS, hPa) and clot stability to lysis (CLS, %).
TM was measured by Enzyme-Linked Immunosorbent Assay (ELISA, Abcam, Cambridge, UK), designed for the quantitative measurement of TM in serum, plasma buffered solutions and supernatants, following protocols from the manufacturer. Normal range lies between 0.625 ng/ml and 20 ng/ml.
ANGPT2 was measured by Enzyme-Linked Immunosorbent Assay (ELISA, Abcam, Cambridge, UK), designed for the quantitative measurement of human ANG2 in serum, plasma buffered solutions and supernatants, following protocols from the manufacturer. Normal range lies between 4.12 pg/ml and 3000 pg/ml
Quantitative determination of plasminogen activator inhibitor-1 (PAI-1) was assessed by Enzyme-Linked Immunosorbent Assay (ELISA, Asserachrom®, Diagnostica Stago, Paris, France) in PPP. Normal range lies between 4 and 43 ng/ml.
The TAFI was determined. On PPP, plasma TAFI is activated with thrombin (Stachrom®, Diagnostica Stago, Paris, France). A partial hydrolysis occurs by activated TAFI of a chromogenic substrate and subsequent hydrolysis by carboxypeptidase A, which entails its discoloration. The measurement of this discoloration is proportional to the TAFI activity in the sample. Normal range lies between 108 ± 24.5%.
Plasma ADAMTS13 activity and VWF antigen were measured using chemiluminescence (AcuStar, International Laboratory, Bedford, MA, USA) following the protocols from the manufacturer. ADAMTS13 is a metalloprotease that cleaves von Willebrand factor, a large protein involved in thrombus formation.