Whole-Blood Thrombin Generation Monitored with a Calibrated Automated Thrombogram-Based Assay

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2012-08, Vol.58 (8), p.1252-1259
• Main Authors: NINIVAGGI, Marisa, APITZ-CASTRO, Rafael, DARGAUD, Yesim, DE LAAT, Bas, HEMKER, H. Coenraad, LINDHOUT, Theo
• Format: Article
• Language: English
• Subjects: Adult; Analytical, structural and metabolic biochemistry; Autoanalysis; Automation; Biological and medical sciences; Blood; Blood platelets; Calibration; Factor VIII - analysis; Fluorescence; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Hematologic Tests - methods; Hemophilia; Hemophilia A - blood; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Mineral oils; Molecular biophysics; Physiology; Plasma; Rhodamines; Sensitivity and Specificity; Thrombin - analysis; Thrombin - biosynthesis
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2012.184077

The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. Thin-layer technology and the use of a rhodamine 110-based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P < 0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma.