title: Can the diagnostic reliability of the thrombin generation test as a global haemostasis assay be improved? The impact of calcium chloride concentration.
abstract: BACKGROUND
  Thrombin generation test (TGT) is a global haemostasis assay with a potential to predict bleeding tendencies and treatment effects in patients with haemophilia. Despite 15 years of clinical research, the diagnostic value of TGT remains controversial, possibly due to suboptimal sensitivity to coagulation deficiencies, robustness and reproducibility.
  
  
  OBJECTIVE
  The goal of this study was to explore the effect of calcium chloride (CaCl2) concentration on the TGT's response to intrinsic coagulation factors (F) VIII, IX and XIa.
  
  
  METHODS
  Normal and factor-deficient plasmas supplemented with lacking coagulation factor and different CaCl2levels were tested by calibrated thrombinography assay.
  
  
  RESULTS
  Thrombin peak height (TPH) was strongly CaCl2dependent, increasing sharply from no TG at 5 mm to a peak at 13.8 mm of CaCl2(95% confidence interval [CI]: 13.0, 14.5) in normal and normalized deficient plasmas and at 11.9 mm (CI: 9.7, 14.2) in deficient plasmas, and then decreasing slowly to a complete inhibition at 30-40 mm. In contrast, TG lag time, time to peak and endogenous thrombin potential were nearly insensitive to CaCl2concentrations between 10 and 20 mm. The maximal difference between the TPH in deficient and supplemented plasmas was observed at 15.5 mm (CI: 12.8, 18.1).
  
  
  CONCLUSIONS
  Variations in CaCl2concentration in the assay mixture and sodium citrate concentrations in patient plasma samples may affect TGT responses, sensitivity and result in increased inter- and intra-laboratory variance. Implementation of TGT by clinical and quality control laboratories may require optimization of CaCl2concentration.
authors.affiliation: Office of Tissues and Advanced Therapies, CBER, US Food and Drug Administration, Silver Spring, MD, USA.|Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russia.
authors.forename: L A
authors.identifier: http://orcid.org/0000-0002-8892-8908
authors.initials: LA
authors.lastname: Parunov
authors.affiliation: Office of Tissues and Advanced Therapies, CBER, US Food and Drug Administration, Silver Spring, MD, USA.|Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russia.
authors.forename: S S
authors.initials: SS
authors.lastname: Surov
authors.affiliation: Office of Tissues and Advanced Therapies, CBER, US Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Office of Tissues and Advanced Therapies, CBER, US Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Lee
authors.affiliation: Office of Tissues and Advanced Therapies, CBER, US Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: M V
authors.identifier: http://orcid.org/0000-0003-2509-1781
authors.initials: MV
authors.lastname: Ovanesov
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002122:Calcium Chloride / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006467:Hemophilia A / Q000150:complications; D006470:Hemorrhage / Q000097:blood / Q000150:complications / Q000175:diagnosis*; D006487:Hemostasis / Q000187:drug effects*; D013917:Thrombin / Q000096:biosynthesis* / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D013925:Thromboplastin; D013917:Thrombin; D002122:Calcium Chloride
keywords: blood coagulation; calcium; calibrated automated thrombogram; haemophilia; quality control; thrombin generation