Novel clinical and diagnostic aspects of antineutrophil cytoplasmic antibodies

Abstract

Antineutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of some idiopathic systemic vasculitides. Besides the investigation of ANCA-associated vasculitis (AAV) and constant effort for a standardized nomenclature and classification of the AAV, a main focus of research during the last few years has been to constantly improve the performance of enzyme immunoassays. With the latest so called third generation ELISA, this goal seemed to be fulfilled. The International Consensus Statement on Testing and Reporting of ANCA gave recommendations for standardized strategies for the serological diagnosis of ANCA. New developments now target the system immanent drawbacks of the respective diagnostic methods, be it the need for batching and the long time to result for ELISA, or the high likelihood of error and subjectivity of indirect immunofluorescence (IIF). Random access technology and multiplexing for solid phase assays as well as digital imaging for IIF are tools which may help to expedite and simplify routine diagnostics in the lab and in emergency settings. Recent findings indicate that PR3-ANCA have clinical utility beyond the diagnosis of AAV. PR3-ANCA can also serve as an aid for the differentiation between ulcerative colitis (UC) and Crohn's disease (CrD) and the stratification of UC patients. This review provides a detailed review of what is known about ANCA and highlights the latest research and state-of-the-art developments in this area.

Figure 1

Appearance of cytoplasmic indirect immunofluorescence pattern (C-ANCA, Figure 1(a)) and perinuclear (P-ANCA, Figure 1(b)) on ethanol-fixed human neutrophil cells. The C-ANCA pattern is largely caused by autoantibodies targeting serine protease proteinase-3 (PR3-ANCA), while the P-ANCA pattern is caused by antibodies binding to many antigens among which myeloperoxidase (MPO-ANCA) is the most frequent target in primary systemic vasculitis [11].

Figure 2

Disease nomenclature system adopted by the 2012 International Chapel Hill Consensus Conference [17].

Figure 3

Representative screenshot of a Multianalyte Screen (i.e., QUANTA Link, INOVA Diagnostics Inc.) showing a C-ANCA and speckled ANA double positive sample. Images obtained on ethanol- and formalin-fixed substrates can be displayed next to Hep-2 ANA results simultaneously and thus allow for interpretation with high accuracy.

Figure 4

Principle of QUANTA-Flash chemiluminescent immunoassays. Paramagnetic beads are coupled with native PR3 or MPO. The beads are then incubated with diluted patient samples. After 9.5 min incubation unbound antibodies are removed by washing. Antihuman IgG isoluminol conjugate (Tracer) is added and binds immobilized antibodies. After another 9.5 min incubation unbound Tracer is removed by washing. Finally, Trigger 1 and Trigger 2 are injected and emerging light is measured. After injection of Trigger 1 and Trigger 2, the luminescence is measured as relative luminescence units (RLU) [17].

Figure 5

Receiver operation characteristics (ROC) analysis of 292 GPA serum samples tested together with 1356 disease controls using a novel PR3 CLIA revealed an area under the curve (AUC) value of 0.78 (95% CI 0.74–0.83), resulting in a clinical sensitivity and specificity of 62.7% (95% CI 56.8–68.2%) and 98.0% (95% CI 97.1–98.7%), respectively.

Figure 6

Comparative receiver operation characteristics (ROC) analysis of 20 P-ANCA positive serum samples tested by IIF together with 75 disease and control serum samples using a novel MPO CLIA and three other commercially available MPO assays revealed AUC values of 0.97 (Wieslab MPO-ANCA), 0.96 (QUANTA Flash MPO), 0.94 (Zenit RA MPO), and 0.94 (ELIA MPO^s) resulting in a relative sensitivity/specificity of 95%/96% (Wieslab MPO), 95%/96% (QUANTA Flash MPO), 86.7%/95.7% (Zenit RA MPO), and 95%/94.7% (ELIA MPO^s) against a positive P-ANCA result by IFA, respectively [18].

Figure 7

Differentiation between ulcerative colitis (UC) and Crohn's disease (CrD). The clinical difference of UC and CrD and the potential role of PR3-ANCA as a marker to help in the diagnosis of UC are illustrated.