Background: Diagnosis of immune-mediated neutropenia is challenging. Flow cytometry has been used to detect neutrophil surface-associated antibody (NSA). However, high NSA percentages in healthy dog control samples resulted in test discontinuation.
Objectives: 1) Retrospectively compare NSA in controls versus patients; 2) prospectively assess NSA in healthy dogs to determine whether Fc block or Fc-specific antibody improves test performance. Animals: 30 paired canine control and patient samples; 10 research Beagles.
Methods: Percent NSA was retrospectively collected from control and patient sample data (6/2023-1/2024). NSA was prospectively quantified by direct immunofluorescence flow cytometry on canine whole blood using the same polyclonal goat anti-dog IgG- and IgM-FITC antibodies as retrospective data. Percent NSA-positive cells were quantified with and without Fc block (n=10). An Fc-specific anti-dog IgG antibody was tested (n=6). Data presented as median (range) with nonparametric statistical tests.
Results: Retrospective control samples had variable NSA [IgG: 97% (17-99%); IgM: 93% (48-99%)] similar to patient samples [IgG: 97% (17-99%); IgM: 87%, (34-99%)] (IgG p=0.8; IgM p=0.05). In prospective samples, NSA detection with Fc block [IgG: 98% (77-99%); IgM: 84% (55-97)] was similar to without Fc block [IgG: 97% (83-99%); IgM: 80% (58-99%)] (IgG p=0.7; IgM p=0.7). Fc-specific anti-dog IgG significantly reduced NSA detection [35% (16-61%)] compared to polyclonal anti-IgG (p=0.001). Conclusions and Clinical Importance: Polyclonal antibodies yield unacceptably high NSA detection, possibly due to native Ig bound to Fc receptors. Fc-specific anti-IgG improves test performance but has not been validated in neutropenic dogs. An alternative IgM antibody has not been identified.
Learning Objectives:
describe the intended clinicla utility of the neutrophil sufrace-associated antibody test.
List two potential reasons for non-specific or off-target antibody binding.
