thritis according to the definition outlined by the ACR(3,4).

Ours was clearly a preliminary study to outline thepotential benefits of targeted US of a single joint. Ourpatient selection reflected the reality that patients tend topresent to rheumatology clinics at variable time points intheir disease course, although we recognize that prespeci-fied limits on disease duration would have been ideal. Weindicated in our discussion that larger studies in this areawould be of interest.

The purpose of our study was to determine the existenceof erosions of the 5th MTP joint only, using US in clinicalsituations where no other major prognostic measure isavailable and where time constraints are present, such asin a busy clinic setting. It was not intended to be a com-prehensive evaluation of US nor a detailed analysis of allsynovial joints. Equally, the study was not designed tomeasure subsequent clinical outcomes. It is known thaterosive disease is associated with a poorer prognosis, as itproves the propensity for joint damage (5). Early determi-nation of joint erosion is therefore useful in planning treat-ment, and the ability to do this in a timely manner is ofvalue in clinical decision making.

With regard to the specific questions relating to our UAgroup, 23% of the patients fulfilled criteria for a diagnosisof RA within 12 months of study inclusion, and only 1 ofthese patients had erosions on US at baseline. Althoughfollowup was limited to 12 months, it is likely that withadditional time more of these UA patients may haveevolved into RA (6). Of the 3 patients in the UA cohortwho subsequently fulfilled criteria for a diagnosis of RA, 1was positive for both anti-CCP antibody and rheumatoidfactor in the absence of US erosions, 1 was positive foranti-CCP antibody alone, and the final patient, althoughnegative for both serologic markers, had US erosion atbaseline.

This study supports the use of US in the diagnosis ofearly erosive disease and lays the foundation for a longi-tudinal study to define the prognostic capability of tar-geted US in patients who present with inflammatory ar-thritis.

Barry J. Sheane, MB, MRCPIGaye Cunnane, MB, PhD, FRCPISt. James’s HospitalDublin, Ireland

1. Verpoort KN, van Dongen H, Allaart CF, Toes RE, BreedveldFC, Huizinga TW. Undifferentiated arthritis: disease courseassessed in several inception cohorts. Clin Exp Rheumatol2004;22 Suppl 35:S12–7.

2. Sokka T. Early rheumatoid arthritis in Finland. Clin Exp Rheu-matol 2003;21(Suppl 31):S133–7.

3. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,Cooper NS, et al. The American Rheumatism Association 1987revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum 1988;31:315–24.

4. Jordan JM. Epidemiology and classification of osteoarthritis. In:Hochberg MC, Silman A, Smolen J, Weinblatt M, Weisman M,editors. Rheumatology. Philadelphia: Elsevier; 2007. p. 1691–703.

5. Odegard S, Landewe R, van der Heijde D, Kvien TK, Mow-inckel P, Uhligi T. Association of early radiographic damagewith impaired physical function in rheumatoid arthritis: a

ten-year, longitudinal observational study in 238 patients. Ar-thritis Rheum 2006;54:68–75.

6. Van Gaalen FA, Linn-Rasker SP, van Venrooij WJ, de Jong BA,Breedveld FC, Verweij CL, et al. Autoantibodies to cyclic cit-rullinated peptides predict progression to rheumatoid arthritisin patients with undifferentiated arthritis: a prospective cohortstudy. Arthritis Rheum 2004;50:709–15.

DOI 10.1002/acr.20144

Association of inflammatory mediatorswith premature atherosclerosis inrheumatoid arthritis and healthy controls:comment on the article by Rho et al

To the Editors:

We read with interest the article by Rho et al, publishedrecently in Arthritis Care & Research (1), reporting thatinflammatory markers interleukin-6 (IL-6), tumor necrosisfactor � (TNF�), and serum amyloid A (SAA) had positiveassociations with coronary artery calcium (CAC) in rheu-matoid arthritis (RA) patients (n � 169), but had border-line “inverse” associations among controls (n � 92). Theseresults among controls are somewhat surprising. Majorclinical end point studies indicate that IL-6 is positivelyassociated with cardiovascular disease (CVD) risk inde-pendent of classic risk factors in cohorts without RA (2),while elevated SAA and TNF� levels often show, at theleast, trends toward being associated with increased CVDrisk (3,4). Indeed, a recent large study in healthy peopledemonstrated weak positive (although not independent)associations of IL-6 with CAC (5). Furthermore, in thestudy by Rho et al, serum levels of at least some of theinflammatory markers in healthy controls are somewhathigher than might be expected from prior literature (6,7).Such differences in absolute levels may be due partially toassay differences (e.g., detection antibodies) and lack ofinternational standards. However, it is also worth consid-ering the relatively poor reproducibility (and possiblelower sensitivity) of multiplex assays. Rho et al reportinterassay coefficients of variation (CVs) ranging from 6.8–21%, but they do not note individual biomarker CVs, orwhether the reported CVs are their own or the manufac-turer’s. Finally, there are emerging concerns that somemultiplex assays may have interference from rheumatoidfactor (RF) (8,9), an issue that requires further explorationin all multiplex assays.

We appreciate that Rho and colleagues refrain in thearticle discussion from overinterpreting the data obtainedfrom their small control population with very little prev-alent CAC (therefore, low power). However, due to theabove issues, we suggest that their apparent “interaction”between RA patients and controls in the association ofinflammatory markers with CAC may be spurious. Untilfurther methodologic research into multiplex analysis foruse in epidemiologic research is undertaken, particularlyin studies comprising RA populations, caution should beused when interpreting data from multiplex-based studies.

1052 Letters

This is particularly true when such studies generate re-sults that conflict with much of the rest of the literature.

Paul Welsh, PhDNaveed Sattar, MD, PhDUniversity of GlasgowGlasgow, UKMike Peters, MD, PhDVU University Medical CenterAmsterdam, The Netherlands

1. Rho YH, Chung CP, Oeser A, Solus J, Asanuma Y, Sokka T, etal. Inflammatory mediators and premature coronary atheroscle-rosis in rheumatoid arthritis. Arthritis Rheum 2009;61:1580–5.

2. Sattar N, Murray HM, Welsh P, Blauw GJ, Buckley BM, CobbeS, et al. Are markers of inflammation more strongly associatedwith risk for fatal than for nonfatal vascular events? PLoS Med2009;6:e1000099.

3. Kip KE, Marroquin OC, Shaw LJ, Arant CB, Wessel TR, OlsonMB, et al. Global inflammation predicts cardiovascular risk inwomen: a report from the Women’s Ischemia Syndrome Eval-uation (WISE) study. Am Heart J 2005;150:900–6.

4. Jefferis BJ, Whincup PH, Welsh P, Wannamethee SG, RumleyA, Lennon LT, et al. Circulating TNF� levels in older men andwomen do not show independent prospective relations withMI or stroke. Atherosclerosis 2009;205:302–8.

5. Jenny NS, Brown ER, Detrano R, Folsom AR, Saad MF, Shea S,et al. Associations of inflammatory markers with coronary ar-tery calcification: results from the Multi-Ethnic Study of Ath-erosclerosis. Atherosclerosis 2010;209:226–9.

6. Sattar N, Murray HM, Welsh P, Blauw GJ, Buckley BM, deCraen AJ, et al. Are elevated circulating intercellular adhesionmolecule 1 levels more strongly predictive of diabetes thanvascular risk? Outcome of a prospective study in the elderly.Diabetologia 2009;52:235–9.

7. Welsh P, Woodward M, Rumley A, Lowe G. Associations ofplasma pro-inflammatory cytokines, fibrinogen, viscosity andC-reactive protein with cardiovascular risk factors and socialdeprivation: the fourth Glasgow MONICA study. Br J Haematol2008;141:852–61.

8. De Jager W, Rijkers GT. Solid-phase and bead-based cytokineimmunoassay: a comparison. Methods 2006;38:294–303.

9. Todd DJ, Knowlton N, Karlson EW, Shadick NA, WeinblattME, Schur PH, et al. Rheumatoid factor interferes with multi-plex-based ELISA in patients with rheumatoid arthritis [ab-stract]. Arthritis Rheum 2009;60 Suppl:S279.

DOI 10.1002/acr.20158

Reply

To the Editors:

We thank Dr. Welsh and colleagues for their thoughtfulcomments. They suggest that concentrations of some in-flammatory markers were higher in our control subjectsthan might be expected from published examples thatreport intercellular adhesion molecule 1 (ICAM-1) (1),IL-6, and TNF� concentrations (2). We found that ICAM-1concentrations (median 119.4, interquartile range [IQR]98.3–161.8 ng/ml) appeared to be somewhat lower, ratherthan higher, than those reported by Sattar et al (geometricmean � SD 369.4 � 1.39 ng/ml) (1). TNF� concentrationsin our control subjects (median 3.4, IQR 2.4–4.8 pg/ml)were similar to those reported by Schaap et al in theDynamics of Health, Aging, and Body Composition Studyin an elderly population of 1,023 men (median 3.21, IQR2.43–4.07 pg/ml) and 1,154 women (median 3.04, IQR

2.35–3.84 pg/ml) (3). Concentrations of IL-6 in our controls(median 4.2, IQR 1.2–18.2 pg/ml) were indeed somewhathigher than those generally reported in the literature, butwere similar to those reported in the top one-third ofcontrol subjects in a large epidemiologic study (4) wherethe range was 2.5–28.1 pg/ml with a geometric mean of 4.2pg/ml. Differences in median values among studies arelikely to be due to relative differences resulting from vari-ation in assay and subject characteristics, rather than noisein the assays, which would bias comparisons betweengroups in a study towards the null.

We agree that enzyme-linked immunosorbent assays(ELISAs) have inherent methodologic limitations, but theseare not limited to multiplex assays. We did not set out tocharacterize the sensitivity and specificity of the assay,and the range of interassay CVs for the cytokine assaysreported in our article (6.8–21%) were those of the man-ufacturer. Intra-assay reproducibility was good. For exam-ple, we found that for IL-6 the average percentage differ-ence between 221 pairs of duplicate samples was 4.0%(95% confidence interval [95% CI] �0.87%, 9.4%); using aBland-Altman plot analysis (5), the mean absolute differ-ence between 2 duplicate samples was 0.96 (95% CI 0.90,1.01 pg/ml).

Circulating antibodies such as RF that are present inserum could affect immunodetection assays; therefore,commercially available ELISAs include proprietary block-ing reagents to minimize heterophilic interference. Never-theless, we cannot exclude the possibility of interferenceby RF in the assay. However, there was no evidence of asystematic effect (an increase in all cytokines) in samplesfrom patients with RF since only 2 (IL-6 and TNF�) of the8 inflammatory mediators measured using the multiplexELISA were significantly higher in patients with RF. Anincrease in these 2 cytokines in patients with RF is notunexpected given the association between RF and moresevere disease. Furthermore, when we restricted our mainanalysis to only patients with RA and further adjusted thestatistical model for RF (present or absent), TNF� (P �0.002) and IL-6 (P � 0.026) remained significantly associ-ated with coronary calcification.

As Welsh and colleagues mention, we did not set out todefine the determinants of coronary calcification in controlsubjects since other studies with larger samples andgreater power have done this. The borderline significantnegative association between IL-6 concentrations and cor-onary calcification in control subjects was surprising, andwe deliberately did not overinterpret this finding given thesmall number of control subjects with coronary calcifica-tion (n � 35). However, the significance of the positiveassociations between IL-6 and TNF� concentrations andcoronary calcification in patients with RA was not depen-dent on the findings in control subjects, as shown above.Given the limitations of the study, we agree that the P valuefor interaction between disease status and mediator andcoronary calcification should be interpreted cautiously.However, the relationship between these cytokines andcoronary calcification in patients with RA is clear.

Our novel finding that IL-6 and TNF� were associatedwith coronary artery calcification in RA is concordant with

Letters 1053