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We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routine venipuncture into tubes containing heparin as anticoagulant (but not citrate, acid-citrate-dextrose, EDTA, or oxalate) and if plasma antithrombin III concentration was > or = 30% of normal. Heparinized plasma F1.2 was stable for > or = 8 h at 20-25 degrees C, and if premixed with a stabilizing reagent, for > or = 4 years at -70 degrees C. Mean values for heparinized plasma F1.2 collected and stored by recommended procedures were increased in patients with thrombosis and conditions of increased thrombotic risk, and were sensitive to heparin and oral anticoagulant therapies. We conclude that plasma obtained by routine venipuncture into tubes with heparin as anticoagulant is an appropriate specimen for F1.2 measurements for most patients.

Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

Prothrombin fragment 1.2 (F1.2) is a biomarker of thrombin generation during blood coagulation and has diagnostic potential for assessing thrombotic risk and monitoring anticoagulation therapy. We used a monoclonal antibody-based immunoassay for plasma F1.2 to establish a well-defined reference interval and to evaluate the effects of age, race, sex, and smoking status on F1.2 concentrations in a healthy population. Plasma samples and demographic information were obtained from 357 healthy individuals. F1.2 concentrations more closely followed a lognormal than a gaussian frequency distribution. In a multiple linear-regression model in which the logarithms of F1.2 concentrations were regressed on age, race, sex, and smoking status, the significant explanatory variables were age and, to a lesser extent, sex and smoking. A segmented "hockey stick" regression model indicated that F1.2 concentrations and age were unrelated for individuals < 44 years old but were positively correlated above that age threshold. The estimated 95% tolerance interval (P = 0.95) for F1.2 in healthy individuals < 44 years old (n = 268) was 0.21-2.78 nmol/L. We conclude that age-matched F1.2 reference intervals may be important for studies evaluating the diagnostic utility of F1.2 measurements, and that the clinical relevance of increased thrombin generation during aging warrants further investigation.