title: Determining the impact of instrument variation and automated software algorithms on the TGT in hemophilia and normalized plasma.
abstract: BACKGROUND
  Despite increasing recognition as a more precise test of in vivo hemostatic conditions, standardization of the thrombin generation test (TGT) continues to hinder its development as routine clinical practice. Prior efforts largely focused on comparing the effects of experimental conditions and different reagents. Commercialized kits, instruments and software have been introduced to calculate the TG curve and its parameters. However, modified versions of the TGT continue to be used worldwide on a variety of microplate reader instruments and processed using individualized algorithms. No prior study has compared the effect of instrument choice and its inherent noise profile on the processing of the TG curve and its common endpoint parameters.
  
  
  METHODS
  Hemophilia A plasma supplemented with buffer or Factor VIII, mimicking hemophilic or normalized samples respectively, was monitored for thrombin generation after activation with TF on six different fluorescent microplate readers. Each instrument was optimized for TGT signal recording prior to testing. An automated software package containing various mathematical algorithms was utilized to compute the TG curves and parameters, and compare different TG processing approaches.
  
  
  RESULTS
  Instruments produced unique noise profiles and end-point parameters that were incomparable in absolute signal terms. Similar relative hemophilic responses were obtained across various instruments when the normalized plasma sample was used as an internal standard. Smoothing algorithms corrected destructive instrument noise.
  
  
  CONCLUSIONS
  Instrument-induced errors from numerical differentiation during TG curve processing cannot be eliminated by external calibrators, and require careful qualification of the instrument and implementation of noise-reducing software algorithms.
authors: Office of Blood Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD, USA.
authors: S A
authors: SA
authors: Woodle
authors: A M
authors: AM
authors: Shibeko
authors: T K
authors: TK
authors: Lee
authors: M V
authors: MV
authors: Ovanesov
mesh_terms: D000465:Algorithms; D001331:Automation / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D001780:Blood Coagulation Tests / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D002138:Calibration; D006467:Hemophilia A / Q000097:blood*; D006801:Humans; D012015:Reference Standards; D012984:Software; D013917:Thrombin / Q000096:biosynthesis*
chemical_list: D013917:Thrombin
keywords: 7-amino-4-methylcoumarin; AMC; AUC; Blood Coagulation; CAT; Deficiency; Factor VIII; HA; Hemophilia; LT; NP; TGT; TPH; TTP; Thrombin; area under the curve; assay; calibrated automated thrombinography; hemophilia A plasma; lagtime; normalized plasma; thrombin generation test; thrombin peak height; thrombin-α2macroglobulin; time to peak; α2MG