Prefixation of neutrophils for neutrophil antibody testing

Objective: The granulocyte immunofluorescence test based on flow cytometry is still the most commonly used method for detecting antineutrophil antibodies. We questioned whether neutrophil prefixation is necessary for this test. Methods: Three pretreatment methods using paraformaldehyde were compared: unfixed, short-fixed (3 minutes 40 seconds), and long-fixed (10 minutes) leukocytes. The mean fluorescence intensity (MFI) of a test sample was divided by the average of negative controls, and this MFI ratio of gated neutrophils or lymphocytes was assessed for its discriminatory value based on the pretreatment method used. Results: In neutrophil antibody positive (Ab⁺) sera (n=7), the MFI ratio of unfixed neutrophils (1.97) was higher than that of short- (1.60, P =0.004) or long- (1.71, P =0.203) fixed neutrophils. In HLA Ab+ sera (n=10), the MFI ratio of short-fixed lymphocytes (8.85) was higher than that of unfixed (6.52, P =0.120) or long-fixed (3.86, P =0.055) lymphocytes. In antinuclear Ab+ sera (n=4), the MFI ratio of unfixed neutrophils (2.04) was higher than that of short- (1.23, P =0.047) or long- (1.14, P =0.044) fixed neutrophils. Conclusion: The benefit of a prefixation step depends on the diagnostic purpose. Unfixed neutrophils are more sensitive, but the prefixed neutrophils are more specific. For the diagnosis of autoimmune neutropenia of childhood, prefixation can be omitted to enhance sensitivity.