Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA).
BACKGROUND
The "International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)" requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time.


OBJECTIVE
To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA).


METHODS
Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO).


RESULTS
IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears.


CONCLUSIONS
Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.
Paspaliaris
B
B
Department of Biochemistry, St Vincent's Hospital, Fitzroy, VIC, Australia.
Pamio
M
M
Savige
J
J
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000097:blood*; D001800:Blood Specimen Collection / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D053146:Myeloblastin; D009504:Neutrophils / Q000276:immunology; D009195:Peroxidase / Q000097:blood; D012697:Serine Endopeptidases / Q000097:blood; D014657:Vasculitis / Q000201:enzymology / Q000276:immunology*
D019268:Antibodies, Antineutrophil Cytoplasmic; D009195:Peroxidase; D012697:Serine Endopeptidases; D053146:Myeloblastin