Were generated upon serial dilution of NHP. Fitting the Log-Log plot of clot PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 time vs FV 1-stage activity demonstrated a strong linear relationship between these variables after regression analysis (Figure 2A; R2 = 0.980). Fitting of the Log-Log plot of the initial rate of clot formation vs FV 1-stage activity also demonstrated a strong linear relationship between these variables after regression analysis (Figure 2B; R2 = 0.983). The PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 relationship of bothclot time and initial rate of clot formation vs FV 1-stage activity remained linear in NHP diluted up to approx 1024-fold. Given that the FV concentration in NHP is approx 12-40nM [11], the results indicate that the assay is sensitive to approximately 24-80pM FV in NHP. Our results indicate that the normal range of FV activity in the FV 1-stage activity assay in 15 healthy controls assayed with the FV microplate assay was approximately (Mean ?Standard deviation; Range): 0.96 ?0.14 U/ml; 0.68-1.11 U/ml. This is similar to the FV activity and range (0.661.14 U/ml) in healthy controls reported by Cutler et al. for the FV 1-stage activity measured with an automated analyzer [19]. Under the conditions of the assay using 40-fold diluted NHP, the intra-assay variability of Phenyl (4-chloro-3-fluorophenyl)carbamate the time, extent, and initial rate of clot formation in the FV 1-stage assay among 6 wells on 8 different days was 3.4 , 4.4 , and 3.1 , respectively. The inter-assay variability of the time, extent, and initial rate of clot formation in the FV 1-stage assay among 6 wells of 8 different experiments on 8 different days was 7.1 , 7.8 , and 9.2 , respectively. Thus, the intra- and inter-assay variability of these three variables in the FV 1-stage assay was at a low and acceptable level.Tilley et al. Thrombosis Journal 2011, 9:11 http://www.thrombosisjournal.com/content/9/1/Page 4 ofconsistent with the results reported by Nesheim et al. using a manual tilt-tube FV assay [8]. The FV 2-stage and total activity were also decreased in the DIC patients on average, by approx 44 and 42 , respectively, from NHP. The initial rates and extents of clot formation in the FV 2stage assay in the DIC patients were not largely different from NHP, and varied on average, by approx 9 and 4 , respectively, from that observed with NHP.Figure 2 Standard curves of time and initial rate of clot formation vs Factor V activity in normal pooled human reference plasma using the FV 1-stage microplate assay. NHP was serially diluted (0- to 1024-fold in HBS) and assayed with the FV 1-stage microplate assay as described in the text. Log-Log plots of the times and initial rates of clot formation vs FV activity in the FV 1-stage microplate assay are shown after linear regression modelling of the results in panels A and B, respectively.The standard curve of clot time vs FV 1-stage activity (Figure 2A) was used to measure the FV activity in nine DIC patient plasmas which were (FV 2-stage activity) or were not (FV 1-stage activity) intentionally activated with thrombin and the results are shown in Table 1. All nine DIC patient plasmas exhibited FV 1-stage activities and initial rates of clot formation that were decreased on average, by approx 54 and 18 , respectively, from NHP. The extents of clot formation in the FV 1-stage 3-Bromo-5-chloro-2-fluoroaniline assay in the DIC patients were not largely different from NHP, and increased on average, by approx 13 from NHP. Activation of NHP with thrombin generated an approximate 8-fold increase in FV 2-stage activity above the FV 1stage activity (Tabl.