© Copyright 2009 by the American Association for Clinical Chemistry γ′ Fibrinogen: Evaluation of...

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© 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 2010 http://www.clinchem.org/cgi/reprint/ 56/5/781 © Copyright 2010 by the American Association for Clinical Chemistry Journal Club Journal Club

Transcript of © Copyright 2009 by the American Association for Clinical Chemistry γ′ Fibrinogen: Evaluation of...

© 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 2010

http://www.clinchem.org/cgi/reprint/56/5/781

© Copyright 2010 by the American Association for Clinical Chemistry

Journal ClubJournal Club

© Copyright 2009 by the American Association for Clinical Chemistry

IntroductionIntroduction

High fibrinogen is a risk factor for CVDMeta-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

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Introduction (cont)Introduction (cont)

' Fibrinogen has an altered ' chainArises 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)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 = 1.82 - 27.7) 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 = 1.01 - 1.52). (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. 0.34 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

QuestionQuestion

Is ' fibrinogen simply a surrogate marker for total fibrinogen?

© Copyright 2009 by the American Association for Clinical Chemistry

ResultsResults

Assay precisionRespective within-run, run-to-run, and total imprecision estimates:

• 13.4%, 28.6%, and 29.1% at 0.127 g/L of ' fibrinogen

• 4.8%, 11.2%, and 11.6% at 0.416 g/L of ' fibrinogen

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont)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

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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.

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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

QuestionQuestion

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 1998-2001 and stored frozen until analysis.

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont)Results (cont)

' fibrinogen concentrations in humansCentral 95th 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)

which of the sets of reference values in slide 11 were derived from long-term frozen banked samples?

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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 0.037 g/L to a high of 1.443 g/L. The 2.5th and 97.5th percentile limits for ' fibrinogen were 0.088 to 0.551 g/L. The median concentration was 0.234 g/L and the mean concentration was 0.255 0.119 g/L (SD).

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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 0.76. A maximum diagnostic accuracy of 0.78 was found at a decision threshold of 0.30 g/L.

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Table 1. Association of ' Fibrinogen with Traditional Cardiovascular Risk Factors. aResults are unadjusted mean (1 standard deviation) or %. bP 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.)

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Association of ' Fibrinogen with Traditional Cardiovascular Risk FactorsFactors showing significant positive association

Age, gender, BMI, smoking, diabetes, glucose, and triglycerides

Factors showing significant inverse correlationHDL 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

QuestionQuestion

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

ConclusionsConclusions

The ' fibrinogen ELISA has within-run CVs of 13.4% at 0.127 g/L and 4.8% at 0.416 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