The PR3 test is a chemiluminescent immunoassay (CLIA), for use on IDS automated analyzers. It is used for the quantitative determination of the specific IgG antibodies directed against proteinase 3 (PR3), in human samples of serum or plasma (EDTA and Heparin).

Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against antigens contained in the cytoplasm of granulocytes and monocytes.(1) The ANCAs are serologic markers for several necrotizing vasculitides affecting small- and medium-sized blood vessels; in particular, Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA), pauci-immune necrotizing glomerulonephritis (PING, a form of MPA affecting only the kidney) with extracapillary proliferation, and, to a lesser degree, Churg-Strauss syndrome (CSS), which are often collectively defined as ANCA-associated vasculitides (AAVs).(2)

Much clinical and experimental evidence also suggests that the ANCAs may play a pathogenetic role in AAVs; this is particularly true of ANCA-MPO.(3)

Two principal fluoroscopic classes, c-ANCA and p-ANCA, are susceptible to recognition via the standard indirect immunofluorescence (IIF) method applied to normal human neutrophils fixed with absolute ethanol.  Less frequently, two other fluoroscopic patterns, atypical c-ANCA and ANCA-At, not generally associated with the presence of idiopathic vasculitis, may be recognized by this technique.(4)

In patients affected with AAV, the main antigen targets of the ANCAs are myeloperoxidase (MPO) and proteinase 3 (PR3).(5,6)

MPO is an enzyme with significant bactericide properties that act by catalyzing peroxidation reactions that form toxic products such as HOCl, H2O2, and oxygen radicals. Additionally, hypochlorous acid and its metabolites can inactivate the protease inhibitors and therefore play an important role in tissue degradation and in maintenance of an “inflammatory” microenvironment.(3) MPO accounts for about 5% of the total protein content of the neurophils; it is a strongly cationic dimeric ester molecule with a molecular weight ~ 140 kDa.

PR3 is a weakly cationic serine protease contained in the primary granules (or azurophils) of granulocytes and monocytes, made up of 228 amino acids and having a molecular weight of 29-31 kDa, that exercises an anti-microbial action against bacteria and fungi. The majority of its biological functions depend on proteolytic activity. In an inflammatory context, it is released, together with other constituents of the granules and oxygen radicals, to the exterior of the cell where it can degrade collagen, the proteoglycans, and other constituents of connective tissue. Excessive, prolonged, or inappropriate proteolytic activity, however, is a cause of damage to the organism.(3) PR3-ANCAs interfere with enzymatic inhibition of PR3 by its physiological inhibitor, α1-antitrypsin.

In the majority of cases, a c-ANCA pattern is attributable to the presence of antibodies specific for PR3, while a p-ANCA pattern can be caused by antibodies directed against any of numerous proteins, among which MPO is the most frequent. To date, the clinical significance of ANCAs directed against other cytoplasmic components different from PR3 and MPO is not clear and for this reason they are not useful in laboratory diagnosis of the AAVs.(7,8)

Following publication of the results of the multi-center project for standardization of ANCA assay methods,(9) official guidelines for correct ANCA testing and reporting in patients with suspected vasculitides were drawn up; the reader is referred to said documents for more detailed information (4,10,11). The above-mentioned study, the results of which were later amply confirmed, clearly showed that correct ANCA determination depends on a combination of the IIF method and identification of antigen specificity using systems specific for the two principal antigens, MPO and PR3 (ELISA, FEIA, CLIA methods).(12,13)

Combined use of the two methods permits obtaining a specificity > 98% even in the case of the control pathologies, a percentage much greater than that obtainable when the tests are used singly.

The ANCA titer correlates (although with some exceptions) with disease activity; for this reason, quantitative methods for ANCA-MPO or ANCA-PR3 assay are recommended for use in patient monitoring.(14)

The specificity of the ANCAs (MPO or PR3) does not allow differentiating among the various AAVs (WG, MPA, PING, CSS); nevertheless, the presence of p-ANCA/MPO-ANCA is more suggestive of a diagnosis of MPA or PING, while c-ANCA/PR3-ANCA positivity is more frequently associated with WG.

In an active phase of WG and in MPA (including the kidney-restricted form), the prevalence of the ANCAs is  70-90%; in CSS, the ANCAs are instead present only in ≈ 40% of patients, with MPO as the prevalent antigen specificity.

Regarding the diagnostic utility of the laboratory datum, it is worthwhile noting that the positive and negative predictive values (PPV, NPV) depend, besides on the sensitivity and the specificity of the test, on the prevalence of the disease in the study population. An appropriate requirement (high pre-testing probability)  permits obtaining a result with real clinical utility and significantly reduces the incidence of false positive results.(15)

The ANCA assay should therefore be conducted when there is a founded clinical suspicion; a positive result, while not per se sufficient for diagnosing AAV, should be evaluated in the light of the overall clinical and histologic picture.

Literature occasionally reports ANCA positivity in cases of pathologies different from ANCA-associated vasculitides, although these reports have not always been confirmed by demonstration of antigen specificity for MPO or PR3.

Differential diagnosis must consider numerous pathologies; among these, it is worthwhile to recall – due in part to their high frequency – the infectious diseases and subacute bacterial endocarditis in particular. Despite the fact that the NPV of a negative result is generally elevated, this fact does not completely exclude the possibility of an AAV, above all in patients showing strong clinical signs of primitive systemic vasculitis.(16)

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  2. Jennette JC, Falk RJ. Antineutrophil cytoplasmic autoantibodies and associated diseases : a review. Am J Kidney Dis 1990; 15 : 517-29.
  1. Chen M, Kallenberg CGM. New advantages in the pathogenesis of ANCA-associated vasculitides. Clin Exp Rheumatol 2009, 27 (s. 52 ): 108-14. Review.
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