antihuman IgG. This anti-immunoglobulin probe binds to any human IgG ANA on the slide. The slide is washed again to remove unbound antiimmunoglobulin. Fluorescence of the nuclear structures of the leukocytes indicates an adherence of patient antibodies to nuclear proteins [29].

ANA titers and patterns acquired from different laboratories vary greatly. The factors that lead to variable results include the use of different substrate cells (such as mouse liver, mouse kidney, monkey kidney [Vero cells]) or human tissue culture lines (WIL-2, KB, or Hep-2 cells), different ßuoresceinated antibodies, microscopes of different powers and sensitivities, and varying levels of technical skill. Within the active, untreated SLE patients, positive ANAs are detected in 95% of the tests performed with mouse liver or kidney cells and in 98% of the tests performed with human tissue culture lines. The difference between the two substrates is due, at least in part, to the relative absence of the Ro/SSA antigen in mouse liver or kidney cells and its presence in the tissue culture lines. We advocate ANA testing on two cell lines: one on mouse liver, mouse kidney, or monkey kidney cells, and the other on WIL-2, KB, or Hep-2 cells.

Absence of a positive ANA in tissue culture lines makes the diagnosis of SLE unlikely. A negative ANA in a patient with clinical evidence for a speciÞc connective tissue disease suggests that the test should be repeated for conÞrmation because the result could be due to faulty testing; in case of conÞrmed negative ANA, sequential testing is advocated.

A positive ANA in a patient with clinical evidence for a speciÞc connective tissue disease requires speciÞc ANA testing. A positive ANA in a patient with limited or nonspeciÞc clinical Þndings requires continued observation until disease expression is more complete.

3.2.1.4 Antineutrophil Cytoplasmic Antibodies

Autoantibodies (IgG) directed against a cytoplasmic antigen of human neutrophilsÑthe antineutrophilcytoplasmicantibodies(ANCAs;synonym, anticytoplasmic antibodies or ACPAs)Ñcan be

Table 3.10 Antineutrophil cytoplasmic antibodies (ANCAs): speciÞcities and diseases

c-ANCA (%) p-ANCA (%)

c-ANCA Cytoplasmic pattern, antigen speciÞcity for pro- teinase-3 (PR-3)

p-ANCA Perinuclear pattern, antigen speciÞcity for myeloperoxidase (MPO)

detected in patients with systemic vasculitis, such as granulomatosis with polyangiitis (Wegener), microscopic polyarteritis nodosa, and segmental necrotizing glomerulonephritis [37Ð40]. Although still a subject of debate, these diseases are thought to be part of the spectrum of one disease process.

The presence of ANCAs is speciÞc and sensitive for granulomatosis with polyangiitis (Wegener), an entity often associated with episcleritis and scleritis; the speciÞcity is 99% by indirect immunoßuorescence techniques and 98% by ELISA detection; the sensitivity is 96% for active generalized disease, 67% for active regional disease, and 32% for disease in full remission after initial regional symptoms [38Ð40]. However, the presence of ANCA should be adjunctive and, with very rare exceptions, should not substitute for a tissue diagnosis. False-positive ANCA titers have been reported in certain infectious and neoplastic diseases.

Two types of ANCAs were originally described. A granular cytoplasmic staining pattern produced by c-ANCA is caused by antibody directed in at least 95% of cases against protei- nase-3 (PR-3), a neutral serine protease of human neutrophils [39, 40]. A perinuclear staining pattern produced by p-ANCA is caused by antibodies directed against a variety of different antigens (myeloperoxidase [MPO], elastase, and lactoferrin) [41]. The speciÞcity of a positive PR-3 ANCA (c-ANCA) for granulomatosis with polyangiitis (Wegener) is very high, especially if active glomerulonephritis is present. A small percentage of patients with granulomatosis with polyangiitis (Wegener) may have anti-MPO rather than anti-PR-3 antibodies. The speciÞcity of a positive anti-MPO ANCA (p-ANCA) for