title: Prefixation of neutrophils for neutrophil antibody testing.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Ye Jee
authors.initials: YJ
authors.lastname: Shim
authors.forename: Dong Il
authors.initials: DI
authors.lastname: Won
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000032:analysis* / Q000276:immunology; D000918:Antibody Specificity; D001327:Autoimmune Diseases / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D002675:Child, Preschool; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007223:Infant; D008297:Male; D009503:Neutropenia / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D009504:Neutrophils / Q000276:immunology* / Q000473:pathology; D011108:Polymers / Q000737:chemistry; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; C003043:paraform