© Copyright 2009 by the American Association for Clinical Chemistry γ′ Fibrinogen: Evaluation of a New Assay for Study of Associations with Cardiovascular Disease R.S. Lovely, S.C. Kazmierczak, J.M. Massaro, R.B. D’Agostino Sr., C.J. O’Donnell, and D.H. Farrell May © Copyright 2010 by the American Association for Clinical Chemistry Journal Club

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  High fibrinogen is a risk factor for CVD Meta-analyses show a two times higher risk for each 1 g/L increase  Fibrinogen is heterogeneous with different isoforms ≈ 90% contain only  A chains ≈ 10% contain a  ' chain

© Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)   ' Fibrinogen has an altered  ' chain Arises from alternative mRNA processing Contains a high affinity thrombin binding site Results in altered clot ultrastructure Results in a decreased rate of fibrinolysis

© Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Increased  ' fibrinogen is independently associated with CVD and stroke in case/control studies Coronary artery disease: n = 133, OR = 7.16 (95% CI = ) per  A/  ’ fibrinogen quartile (Lovely RS, Falls LA, Al-Mondhiry HA, Chambers CE, Sexton GJ, Ni H, Farrell DH. Thromb Haemost 2002:88:26-31) Myocardial infarction: n = 774, OR = 1.24 (95% CI = ). (Mannila MN, Lovely RS, Kazmierczak SC, Eriksson P, Samnegård A, Farrell DH, et al. J Thromb Haemost 2007;5:766-73) Stroke: n = 249, elevated in cases vs. controls (0.42 mg/ml vs mg/ml). (Cheung EY, Uitte de Willige S, Vos HL, Leebeek FW, Dippel DW, Bertina RM, de Maat MP. Stroke 2008;39:1033-5)

© Copyright 2009 by the American Association for Clinical Chemistry Question  Is  ' fibrinogen simply a surrogate marker for total fibrinogen?

© Copyright 2009 by the American Association for Clinical Chemistry Results  Assay precision Respective within-run, run-to-run, and total imprecision estimates: 13.4%, 28.6%, and 29.1% at g/L of  ' fibrinogen 4.8%, 11.2%, and 11.6% at g/L of  ' fibrinogen

© Copyright 2009 by the American Association for Clinical Chemistry Results (cont)  Analytical specificity  ' fibrinogen shows 99.9% sequence homology with  A/  A fibrinogen Serial dilutions of anti-  ' monoclonal antibody 2.G2.H9 showed no measurable activity against  A/  A fibrinogen

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. Analytical specificity of 2.G2.H9 towards  ' fibrinogen. The analytical specificity of anti-  ' fibrinogen monoclonal antibody 2.G2.H9 and its ability to differentiate  ' fibrinogen from fibrinogen lacking  ' chains was determined by incubating the indicated dilutions of the antibody with  A/  A or  A/  ' fibrinogen in microtiter wells. Goat anti-mouse IgG/HRP conjugate was added for detection, followed by O- phenylenediamine, and the absorbance was quantitated at 450 nm.

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. Limit of quantification for the  ' fibrinogen ELISA. The limit of quantification of the assay for measurement of  ' fibrinogen concentrations in plasma was determined using 8 separate pools of patient plasma. The limit of quantification for the assay was 0.10 g/L, defined as that concentration of  ' fibrinogen giving a within-run CV of 20% or less.

© Copyright 2009 by the American Association for Clinical Chemistry Question  What are some possible limitations of using long-term frozen banked plasma for assessing reference intervals and diagnostic decision thresholds? The Framingham Offspring cohort samples were collected during the 7th examination cycle from and stored frozen until analysis.

© Copyright 2009 by the American Association for Clinical Chemistry Results (cont)   ' fibrinogen concentrations in humans Central 95 th percentile limits (median value) determined from various studies Framingham offspring controls (n = 2879): 0.088–0.551 g/L (0.234 g/L) Healthy blood donors (n = 120): 0.115–0.460 g/L (0.281 g/L) Control subjects from study on CAD (see ref. 7) (n = 42): 0.125–0.676 g/L (0.242 g/L)

© Copyright 2009 by the American Association for Clinical Chemistry Figure 4.  ' fibrinogen concentrations in healthy individuals from the Framingham Offspring Study.  ' fibrinogen was measured in 2879 participants from the Framingham Offspring Study with no previous history of cardiovascular disease. The reference interval of  ' fibrinogen varied nearly 40-fold, from a low of g/L to a high of g/L. The 2.5 th and 97.5 th percentile limits for  ' fibrinogen were to g/L. The median concentration was g/L and the mean concentration was  g/L (  SD).

© Copyright 2009 by the American Association for Clinical Chemistry Figure 5. Receiver-operating characteristic curve for  ' fibrinogen in CAD patients.  ' fibrinogen concentrations were measured in a prior study in 133 patients referred for elective diagnostic cardiac catheterization. The receiver-operating characteristic curve of  ' fibrinogen concentrations in CAD cases and controls showed an area under the curve of A maximum diagnostic accuracy of 0.78 was found at a decision threshold of 0.30 g/L.

© Copyright 2009 by the American Association for Clinical Chemistry Table 1. Association of  ' Fibrinogen with Traditional Cardiovascular Risk Factors. a Results are unadjusted mean (  1 standard deviation) or %. b P values assess the significance of the difference in mean/% across fibrinogen tertiles and are age and sex-adjusted. (P value for age is sex-adjusted only, and P value for sex is age-adjusted only.)

© Copyright 2009 by the American Association for Clinical Chemistry Association of  ' Fibrinogen with Traditional Cardiovascular Risk Factors  Factors showing significant positive association Age, gender, BMI, smoking, diabetes, glucose, and triglycerides  Factors showing significant inverse correlation HDL cholesterol   ' Fibrinogen does not show significant association with systolic blood pressure and total cholesterol  Total fibrinogen does show a significant association with systolic blood pressure and total cholesterol. Addition of  ' fibrinogen to total cholesterol and hsCRP may provide additional predictive value for assessment of the risk of CVD

© Copyright 2009 by the American Association for Clinical Chemistry Question  What future studies should be performed to further investigate the association between  ' fibrinogen and cardiovascular disease risk?

© Copyright 2009 by the American Association for Clinical Chemistry Conclusions  The  ' fibrinogen ELISA has within-run CVs of 13.4% at g/L and 4.8% at g/L  ROC curve analysis of results from patients with coronary artery disease yielded an area under the curve of 0.76, with a diagnostic accuracy of 0.78 at a decision threshold of 0.30 g/L   ' fibrinogen shows excellent prognostic utility for cardiovascular risk analysis