SUBJECTS AND METHODS

Aged people, patients with RA, and control subjects

The Helsinki Aging Study is a population based joint study of general and specialised health care in the City of Helsinki, Finland. In 1990, 629 randomly selected people aged 75, 80, and 85 years underwent a comprehensive clinical study. At entry, participants were examined clinically by a nurse, general practitioner, neurologist, and cardiologist. The clinical examinations have been described in detail earlier.^8,9 The subjects were re-examined in 1993 (clinical examination including echocardiography), in 1995 (home visits), and in 1999 (home visits including tests for cognition). Serum samples of a random 300 people, taken in 1993 and stored at −20°C, were available for the present study. Serum samples of 33 patients with active RA were obtained from Professor Marjatta Leirisalo-Repo, Department of Medicine, Helsinki University Hospital. Serum samples from 67 middle aged blood donors served as controls.

Antifilaggrin antibody (AFA) and RF determination

Filaggrin was purified from human epidermis as previously described.¹⁰ Briefly, proteins extracted from the epidermis were separated by reversed phase high performance liquid chromatography (HPLC). Fractions containing filaggrin, identified using monoclonal AFA, were then subjected to gel filtration HPLC and, finally, to a second reversed phase HPLC step. Amino acid sequencing and mass spectrometry of internal tryptic peptides were used to confirm the identity of the purified protein.

Enzyme linked immunosorbent assay (ELISA) for AFA was performed as described in detail elsewhere.¹⁰ Briefly, microtitre plates (Nunc, Roskilde, Denmark) were coated with filaggrin (3 μg/ml in 50 mM carbonate buffer, pH 9.6) and then coated with 1% human serum albumin. Sera were diluted 1:500 and binding of IgG class antibodies to solid phase filaggrin was demonstrated with alkaline phosphatase conjugated γ chain-specific antihuman IgG antibodies. The cut off level for positivity was set at the 98th centile for middle aged (40–65 years) blood donors. The interassay coefficient of variation of the methods was 3% for a high positive reference serum and 8% for a low positive serum. IgG class antibodies to cyclic citrullinated, filaggrin derived peptide, cfc1-cyc, described by Schellekens et al,⁷ were measured with a commercial kit (RA Immunoscan, Euro-Diagnostica, Malmö, Sweden) according to the manufacturer’s instructions. The limit for positive reactions, suggested by the manufacturer, was 50 arbitrary antibody units.

For the determination of RF, an immunoturbidimetric method described earlier¹¹ was used.

RESULTS

Figure 1 shows the AFA levels in the sera of elderly people. When the cut off level for the positive reaction was set at the 98th centile of healthy controls, a total of 24 AFA positive subjects were noted, one of whom had RA. Thus, there were 23 cases of false positive reactions, corresponding to about 8%. Figure 2 depicts the anti-CCP level in 300 elderly people, 33 selected RA sera, and 67 controls. Only two anti-CCP positive cases were recorded. The one with the highest level had RA; the other had nothing that could explain the positive finding.

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Figure 1

Distribution of IgG class antibodies to native filaggrin purified from human skin in sera from 300 elderly subjects. The 98th centile of normal human sera is given as the cut off level for positivity.

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Figure 2

Distribution of IgG class antibodies to cyclic filaggrin-derived citrullinated peptide (CCP) in sera from 300 elderly people, 33 patients with RA, and 67 normal human sera.

Figure 3 shows the RF results in the sera of the elderly people. Fifty two (17%) of the 300 sera had values exceeding 20 international units (IU)/ml, corresponding to the 95th centile of 400 healthy adults (median age 42 years, range 19–60) analysed earlier with this test.¹¹ When assessed against the 98.5th centile (corresponding to 40 IU/ml) of these normal sera, 23 of the elderly people (8%) were RF positive. Twenty eight of the 33 (85%) RA sera tested positive for RF (data not shown).

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Figure 3

Occurrence of RF in sera from 300 elderly subjects. Cut off limits for positivity, at the 95th and 98.5th centiles of 400 normal human sera, determined in an earlier study,¹¹ are indicated.

DISCUSSION

The prevalence of false positive RF reactions shows a progressive rise with advancing age, doubling during each 20 year period.^12–14 When using sensitive test techniques, the prevalence is about 2% in the age group 15–34 years, 4% in the age group 35–54 years, 8% in the age group 55–74 years, and still higher at more advanced age. The increase is mainly due to IgM RF, with very little change in IgA RF.¹⁴ In line with published reports, the prevalence of false positive RF in the present series of 78–88 years old healthy subjects was 17%.

Our control group for AFA and anti-CCP determinations consisted of blood donors aged 40–65 years (mean 50), selected to represent ages of patients for whom immunological tests for RA are typically performed. When the cut off level for positive reactions in the AFA test was set at the 98th centile of controls, about 8% of the elderly subjects yielded false positive reactions—that is, the prevalence was fourfold compared with the controls. For the determination of AFA, we used filaggrin purified from human skin as an antigen. Sufficient material prepared in this way is not available for large scale testing. The published evidence, however, indicates a high correlation between tests based on filaggrin purified from human skin and on recombinant, properly deiminated filaggrin.¹⁵ Thus, information derived from the former has certain relevance for the latter.

Only a modest correlation existed between tests for AFA and anti-CCP, suggesting that, in part, different antigens elicited the antibody responses.¹⁶ Possibly, antigens in filaggrin recognised by the sera from aged subjects differ from those recognised by sera from patients with RA. Whatever the reason, we conclude that the test for anti-CCP has better specificity than the test for AFA among the aged.