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Ann Rheum Dis. 2007 August; 66(8): 1121–1122.
PMCID: PMC1954712

New markers and an old phenomenon: prozone effect disturbing detection of filaggrin (keratin) autoantibodies

Anti‐filaggrin antibodies, also named anti‐keratin antibodies (AKA), are serum IgG labelling the stratum corneum of the rat oesophagus epithelium, detectable by indirect immunofluorescence (IIF). They constitute a specific marker for the diagnosis of rheumatoid arthritis (RA).1,2,3

A laminar staining pattern is seen with most AKA‐positive sera (fig 1A1A),), but occasionally the cornified layer is diffusely labelled, and the image also shows foamy staining (fig 1B1B).). Although most authors consider sera as AKA positive only when a linear laminated pattern is observed,4,5 some consider the sera as AKA positive even when the staining is diffuse or foamy.6

figure ar66027.f1
Figure 1 Anti‐keratin antibodies (AKA) detected by indirect immunofluorescence on rat oesophagus. (A) Typical linear laminated labelling pattern restricted to the upper cornified layer observed in the serum of patients with rheumatoid ...

On evaluating in our laboratory7 the performance of a second‐generation enzyme immunoassay for detecting anti‐cyclic citrullinated peptide (anti‐CCP) antibodies, directed against the immunogenic target of the AKA,8 we noticed that some of the foamy AKA sera had very high titres of anti‐CCP antibodies.

We then evaluated the anti‐CCP antibodies of all the foamy AKA sera considered as AKA negative in our laboratory from November 2001 to March 2005. AKA were searched with rat oesophagus slides (Biomedical Diagnostics, Ann Arbor, Michigan, USA), sera diluted 1:20, and a rabbit anti‐human fluorescein isothiocyanate‐labelled IgG (Dako SA). Anti‐CCP antibodies were assessed by ELISA (Euroimmun), according to the manufacturer's instructions. Sera were considered positive if the anti‐CCP antibodies were >5 arbitrary units per ml. Rheumatoid factors were also measured by nephelometry on a BNprospec (Dade Behring), using the commercial kit N Latex RF (Dade Behring). Sera were considered as positive for values >10 IU/ml. Clinical diagnoses are given when they are available. Table 11 lists the results. Only four patients were truly positive for AKA without anti‐CCP antibodies (one with polymyalgia rheumatica, one with anti‐phospholipid syndrome and two with uveitis—in one ankylosing spondylitis and in the other sarcoidosis).

Table thumbnail
Table 1 Rheumatological serology of patients with anti‐keratin antibodies with foamy staining from November 2001 to March 2005

Out of 20 sera with foamy staining, 12 were positive for anti‐CCP antibodies and rheumatoid factors with high titres. Only 2 out of 12 patients were not diagnosed with RA, according to the physician. One of the two patients had an uncertain diagnosis: psoriatic arthritis or RA with cutaneous psoriasis (chronic inflammatory rheumatism; patient 8). The other one (patient 3) was an outpatient with no more clinical indication than Sjögren's syndrome associated with vasculitis.

These results reminded us of a very old phenomenon called the prozone effect,9 sometimes described in tests using the IIF technique. An excess of antibodies can result in diffuse or faint staining.10

To confirm this hypothesis, some of the anti‐CCP positive sera, usually diluted 1:20 for AKA determination, were then diluted in phosphate‐buffered saline up to 1:200. Patients 6, 7 and 10 then showed the characteristic linear laminated pattern. These patients who were first considered to be AKA negative were in fact AKA positive!

To conclude, testing for autoantibodies is highly relevant in the diagnosis or exclusion of many systemic autoimmune diseases, including RA. Our data show that IIF is an interesting diagnostic tool, provided the biologist has trained staff, quality controls, proficiency testing and keeps in mind the possible occurrence of prozone effects. As much as possible, AKA and anti‐CCP antibodies should always be combined for optimum diagnostic performance. Solid‐phase assays, which may be too expensive in some countries, can detect only anti‐CCP antibodies, whereas IIF could give extra information such as the presence of antinuclear antibodies when examining for AKA.


AKA - antikeratin antibodies

anti‐CCP - anti‐cyclic citrullinated peptide

IIF - indirect immunofluorescence

RA - rheumatoid arthritis


Competing interests: None declared.


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