A standardized blood test for the routine clinical diagnosis of impaired GM-CSF signaling using flow cytometry

Abstract

Impaired signaling by granulocyte/macrophage-colony stimulating factor (GM-CSF) drives the pathogenesis of two diseases (autoimmune and hereditary pulmonary alveolar proteinosis (PAP)) representing over ninety percent of patients who develop PAP syndrome but not a broad spectrum of diseases that cause PAP by other mechanisms. We previously exploited the ability of GM-CSF to rapidly increase cell-surface CD11b levels on neutrophils (CD11bSurface) to establish the CD11b stimulation index (CD11b-SI), a test enabling the clinical research diagnosis of impaired GM-CSF signaling based on measuring CD11bSurface by flow cytometry using fresh, heparinized blood. (CD11b-SI is defined as GM-CSF-stimulated- CD11bSurface minus unstimulated CD11bSurface divided by un-stimulated CD11bSurface multiplied 100.) Notwithstanding important and unique diagnostic utility, the test is sensitive to experimental conditions that can affect test performance. The present study was undertaken to optimize and standardize CD11b-SI test for detecting impaired GM-CSF signaling in heparinized human blood specimens from PAP patients. Results demonstrated the test was sensitive to choice of anticoagulant, pretesting incubation on ice, a delay between phlebotomy and test performance of more than one hour, and the concentration GM-CSF used to stimulate blood. The standardized CD11b-SI test reliably distinguished blood specimens from autoimmune PAP patients with impaired GM-CSF signaling from those of health people with normal signaling. Intra-subject differences were smaller than inter-subject differences in repeated measures. Receiver operating characteristic curve analysis identified a CD11b-SI test result of 112 as the optimal cut off threshold for diagnosis of impaired GM-CSF signaling in autoimmune PAP for which the sensitivity and specificity were both 100%. These results support the use of this standardized CD11b-SI for routine clinical identification of impaired GM-CSF signaling in patients with autoimmune PAP. The CD11b-SI may also have utility in clinical trials of novel therapeutic strategies targeting reduction in GM-CSF bioactivity now under evaluation for multiple common autoimmune and inflammatory disorders.

Secondary pulmonary alveolar proteinosis after lung transplantation: a single-centre series

“…Diagnosis was delayed in the four case studies and was established only after specific re-examination of the transbronchial biopsies/ BALF. In the autoimmune and genetic forms of PAP [6,9], the alveolar macrophage dysfunction is known to be driven by impaired GM-CSF signalling. A similar impaired GM-CSF pathway was found in one patient by using a recently described test based on the ability of GM-CSF to rapidly increase cell-surface CD11b levels on neutrophils in flow cytometry [6,9].…”

“…Re-examination of transbronchial biopsies from post-operative day 134 (figure 1b) and a new BALF analysis stained with periodic acid-Schiff (PAS) reagent (figure 1c) led to a diagnosis of PAP. Anti-(GM-CSF) antibodies were absent and the possibility of impaired GM-CSF signalling was investigated using a recently described test based on the ability of GM-CSF to rapidly increase cell-surface CD11b levels on neutrophils in flow cytometry (reflected by a CD11b stimulation index) [6]. The GM-CSF stimulation index in patient one was very low, with a value of seven as compared to values of 101, 55 and 102 in three stable lung-transplantation recipients used as positive controls (mean GM-CSF stimulation index of stable lung transplantation=86±26).…”

The Role of GM-CSF Autoantibodies in Infection and Autoimmune Pulmonary Alveolar Proteinosis: A Concise Review

“…The resulting CD11b stimulation index can effectively discriminate between healthy individuals and those with aPAP who exhibit significantly reduced induction of cell surface CD11b expression. This method has a reported sensitivity and specificity of 100%, aiding in the diagnosis of aPAP ( 85 ). Quantification of intracellular phosphorylated STAT5 (pSTAT5) in whole blood or peripheral blood mononuclear cells can similarly discriminate between healthy individuals and those with aPAP ( 8 ).…”

Pulmonary alveolar proteinosis: from classification to therapy

“…This test must be considered a screening test because GM-CSF can be increased during infections in patients without hereditary PAP. GM-CSF signalling can be evaluated by quantifying the level of intracellular phosphorylated STAT5 or cell-surface CD11b in neutrophils in response to GM-CSF [ 32 ]. Positive results require subsequent gene analysis, looking for CSF2RA or CSF2RB mutations.…”