Flow cytometric detection of anti-neutrophil cytoplasmic autoantibodies

An intracytoplasmic immunofluorescence staining technique which allows the detection and quantification of anti-neutrophil cytoplasmic autoantibodies (ANCA) by flow cytometry is described. A polymorph neutrophil population from human peripheral blood was used in this study as indicator cells. These were fixed and permeabilized by paraformaldehyde, Tween 20 and saponin, to allow ANCA in the patients sera to reach their intracellular antigen targets. The numbers of indicator cells remained unaltered by the permeabilization protocol and no cell aggregation or loss of intracellular antigenicity was observed. An excellent agreement (91% (207/228)) between ANCA detection by immunofluorescence microscopy (IF) and flow cytometry was noted. Compared with IF assay, the flow cytometric method has a sensitivity of 93% (42/45) and a specificity of 90% (165/183). Although not able to discriminate between P-ANCA or C-ANCA, this flow cytometric method has the advantage of providing an objective, reproducible and quantitative measure of ANCA, which makes it an ideal technique for screening of patients sera for ANCA reactivities.