Respiratol T Medicine (1992) 86, 367-369

Anti-neutrophil cytoplasmic antibodies Anti-neutrophil cytoplasmic antibodies (ANCA)

were first described in 1982 in patients with necrotising glomerulonephritis (I). In 1985 a Dutch-Danish col- laboration demonstrated that a distinct cytoplasmic fluorescence pattern was associated with Wegener's Granulomatosis (WG) and correlated with disease activity (2); this was later confirmed by other groups (3,4). ANCA testing is now commonplace and may have led to increased diagnostic awareness of Wegener's Granulomatosis and microscopic poly- arteritis (5). Since 1988 there have been three inter- national workshops on ANCA, and there has been great progress in standardizing methodology, identify- ing and cloning the major ANCA antigens, associating ANCA subtypes with clinicopathological syndromes and showing a potential pathogenetic role for ANCA. So, where do we now stand?

Indirect immunofluorescence (IIF) is the most widely used method for detecting ANCA and a standardized methodology has been adopted (6) using alcohol-fixed cells. There are two major patterns: the classical, bright coarsely granular, centrally accen- tuated immunofluorescent staining (cANCA); and perinuclear staining (pANCA). The pANCA pattern is indistinguishable from that produced by granulo- cyte-specific antinuclear antibodies (GS-ANA), but formaldehyde fixation (preventing migration of posi- tively charged basic proteins to the negatively charged nucleus) converts the pANCA, but not the GS-ANA, to cANCA pattern (7). The major cANCA antigen has been identified as proteinase 3 (PR3) and the common pANCA antigen as myeloperoxidase (MPO); both are present in the neutrophil primary granules. Other more rare ANCA antigens include CAP 57, elastase, lactoferrin and cathepsin G; but may sometimes reflect a secondary response to antigens released from neutro- phils as vasculitis progresses (8). Up to 5% ofcANCA react to an as yet unidentified antigen.

Problems with interpretation of I IF patterns prompted development of solid-phase assays. Initially using very crude extracts there are now a number of antigen-specific ELISAs. These tests have both increased our knowledge and added to the confusion. Anti-.MPO ELISA has shown that some MPO-ANCA exhibit a cANCA rather than pANCA IIF pattern, a problem in diagnostic classification if not in treatment of patients. Correlation between IIF and ELISA is generally good but there can be significant differences in titres and occurrence. ELISAs are useful when IIF patterns are atypical or obscured by the presence of simultaneous ANA.

What is the clinical significance of ANCA testing? As for all serological tests ANCA assays have limitations. Even if we assume the ANCA test has 95% nosological specificity and sensitivity [based on Jennette's experience with 3500 assays (9)], then its positive predictive value (diagnostic specificity) in patients with typical clinical and histological findings of active WG will be >95%, and in patients with vasculitis it is still >90%, but in a general hospital population the low prevalence of ANCA-associated disease means that most positive ANCA tests will turn out to be 'false positives'. ANCA tests must therefore be interpreted in the context of other clinical data.

Wegener's Granulomatosis, the most specifically defined disease in the spectrum of ANCA-associated vasculitides, is almost completely confined to the cANCA category, c A N C A - or better, anti-PR3 anti- b o d y - positivity will probably become a third diag- nostic criterion for WG, and has already extended our understanding of the clinical spectrum of disease, especially early forms and variants. However, there are problems in assessing this relationship as WG has protean manifestations and histological proof is often difficult to obtain. Also, about a third of cases present in a grumbling, low-grade or local form and the diag- nostic sensitivity o fcANCA for WG varies according to disease status - in active generalised WG, sensitivity is 93-96%, in active local disease is 67% and in in- active or treated disease is only 37% (10). In these latter cases the cANCA pattern may change to a weakly positive non-specific or pANCA pattern. ELISA sensitivities are a little lower in active and higher in inactive/treated disease. Most quoted diag- nostic specificities of cANCA for WG give a range of 89-99%. However, some other vasculitides can be cANCA positive. Therapeutically this is not as dis- turbing as the cANCA positivity reported in HIV- related diseases (11) and cystic fibrosis patients with vasculitis (12) and with infective episodes (13), although others refute this association with infection (14). Other reports of cANCA appearing in SBE, lymphoma, relapsing polychondritis and bronchial carcinoma highlight the continuing importance of obtaining histological confirmation of diagnosis. Hopefully, in the future, PR3-specific ELISAs will show whether these false positives are of different antigen specificities.

The interpretation of pANCA is less clear, pANCA/ MPO-ANCA is closely associated with a number of vasculitic syndromes, usually small vessel but some median or even large vessel vasculitis. Microscopic

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polyarteritis, systemic potyarteritis nodosa, Churg- Strauss syndrome and various overlap vasculitic syndromes are commonly pANCA, occasionally cANCA, positive. This has been particularly well studied in rapidly progressive glomerulonephritis, where the type of ANCA and anti-GBM antibodies provide better indication of prognosis than the extent of histological changes (15). Ten to fifteen percent of cases with anti-GBM antibodies are also pANCA/ MPO positive. For the respiratory specialist, it is significant that up to 30% of cases with anti-MPO antibody positive renal disease will develop pulmon- ary problems, usually haemorrhage. Within the recognized connective tissue disorders, although the majority of cases will be ANCA negative, a few with rheumatoid arthritis or SLE will show the pANCA IIF pattern, due to the presence of GS-ANA, ANA, MPO and/or elastase antibodies. There is no definite re- lationship with disease activity or vasculitis, although i f A N A and MPO-ANCA are both present the under- lying disease is clinically ANCA rather than ANA related.

An atypical diffuse non-granular cytoplasmic I IF staining pattern creates problems in interpretation. Although sometimes due to poor methodology or weak positive non-specific ANCA, there are con- ditions associated with this appearance-Kawasak i disease (16), ulcerative colitis (17) and rheumatoid a r thr i t i s - all due to antigens as yet unidentified.

In W G it appears that nearly all relapses are preceded or accompanied by a rise in ANCA titres, particularly helpful in differentiating disease relapse from intercurrent infection (4). However, not all rises in ANCA titre are followed by relapse, and treatment regimens based on ANCA titres alone will give some patients unnecessary potent therapy (18). The same is true for other ANCA-associated vasculitides (19). Despite complete clinical remission, ANCA in low titre can be very persistent, particularly using ELISA. In some cases the cause is a switch from high affinity IgG3 subclass antibodies in active disease to low affinity IgG subclasses during remission. Therefore, ANCA testing has much better negative than positive predictive value for disease monitoring.

The role of ANCA in the pathogenesis of vasculi- tides is still uncertain. The presence of ANCA in Rheumatoid, SLE, HIV-related and other conditions suggests they are an epiphenomenon of a more generalized auto-immune disturbance. However, neutrophil invasion and lysis is a characteristic feature of the acute phase of vascular injury in all ANCA- associated vasculitides. In the presence of ANCA there is disturbance and activation of neutrophil function. It is possible that a trigering event such as infection

leading to activation of leucocytes might, in the presence of ANCA, tilt the balance towards frank neutrophil activation and tissue injury. It is interesting that pooled, high-dose intravenous immunoglobulin contains antibodies reactive with ANCA, and has been successfully used to treat systemic vasculitis (20), presumably by suppressing ANCA binding. T-cells are also involved in the pathogenesis of WG and T-cell activation, possibly by cell surface anti-PR3 antigen complexes, has been identified.

In summary, it is to be hoped that standardization of staining methods and interpretation will improve the general reliability of ANCA test r e su l t s - i t will never be perfect. It is still essential to relate the ANCA results to the clinical and, where possible, histological context both in diagnosis and monitoring of disease activity. Not all WG is cANCA positive, because of variation in disease extent and activity, and not all cANCA is WG, but must be considered. There are now a con- siderable number of diseases associated with the increasingly varied subtypes of ANCA and in future the antigen-specific ANCA assays should improve the immunopathological classification of vasculitides.

C. M. B. HIGGS Chest Unit

Royal United Hospital

Bath BA1 3NG

U.K.

References

1. Davies DJ, Moran JE, Niall JF, Ryan GB. Segmental necrotising glomerulonephritis with antineutrophil anti- body: possible arbovirus aetiology? Br Med J 1982; 285: 606.

2. Van der Woude F J, Rasmussen N, Lobatto S et al. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's Granulomatosis. Lancet 1985; i: 425-429.

3. Gross WL, Ludemann J, Kiefer G, Lehmann H. Anti- cytoplasmic antibodies in Wegener's granulomatosis. Lancet 1986; i: 806.

4. Nolle B, Specks U, Ludemann J, Rohrbach MS, DeRemee RA, Gross WL. Anticytoplasmic autoanti- bodies: their immunodiagnostic value in Wegener Granulomatosis. Ann Intern Med 1989; 111: 28-40.

5. Andrews M, Edmunds M, Campbell A, Walls J, Feehally J. Systemic vasculitis in the 1980's - is there an increasing incidence of Wegener's granulomatosis and microscopic polyarteritis? J R Coil Physicians 1990; 24: 284-288.

6. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. Acta Pathol Microbiol lmrnunol Scand 1989; 97 (suppl. 6): 12-I 3.

7. Wieslander J. How are antineutrophil cytoplasmic autoantibodies detected? Am J KidDis 1991; 18:154-I 58.

8. Lesavre P. Antineutrophil cytoplasmic autoantibodies antigen specificity. Am JKidDis 1991; 18: 159-163.

9. Jennette JC. Antineutrophil cytoplasmic autoantibody- associated diseases: a pathologist's perspective. Am J Kid Dis 1991; 18: 164-170.

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10. Cohen Tervaert JW, Van der Woude FJ, Fauci AS et al. Association between active Wegener's granulomatosis and anticytoplasmic antibodies. Arch hltern Med 1989; 149:2461-2465.

I 1. Koderisch J, Andrassy K, Rasmussen N, Hartmann M, Tilgen W. ~False-positive' anti-neutrophil cytoplasmic antibodies in HIV infection. Lancet 1990; 335: 1227-1228.

12. Finnegan M J, Hinchcliffe J, Russel-Jones D et al. Vasculitis complicating cystic fibrosis. Q J Med 1989: 72: 609-62 I.

13. Efthimiou J, Spickett G, Lane D, Thompson A. Anti- neutrophil cytoplasmic antibodies, cystic fibrosis, and infection. Lancet 1991; 337: 1037-1038.

14. Schmitt WH, Csernok E, Gross WL. ANCA and infec- tion. Lancet 1991; 337: 1416-1417.

15. Saxena R, Bygren P, Rasmussen N, Wieslander J. Circulating autoantibodies in patients with extracapill-

ary glomerulonephritis. Nephrol Dial Transplant 1991; 6: 389-397.

16. Savage COS, Tizard J, Jayne D, Lockwood CM, Dillon MJ. Antineutrophil cytoplasmic antibodies in Kawasaki disease. Arch Dis Child 1989; 64: 360-363.

17. Saxon A, Shanahan F, Landers C, Ganz T, Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin lmmuno11990; 86: 202-210.

18. Cohen Tervaert JW, Huitema MG, Hene RJ et al. Prevention of relapses in Wegener's granulomatosis by treatment based on antineutrophil cytoplasmic antibody titre. Lancet 1990; 336:709-711.

19. Savage CO, Lockwood CM. Antineutrophil antibodies in vasculitis. Adv Nephro11990; 19: 225-236.

20. Jayne DR, Davies M J, Fox CJ, Black CM, Lockwood CM. Treatment of systemic vasculitis with pooled intra- venous immunoglobulin. Lancet 1991; 337:1137-1139.