Flow Cytometric Analysis of ALS Patients

Peripheral blood samples from 50 age and gender matched healthy volunteers and 80 ALS patients were collected in K₂EDTA tubes (BectonDickinson) at initial and return visits as indicated. Un-manipulated whole blood was stained directly with antibodies. For a subset of initial patients (n = 17) flow cytometry was performed on the 2 laser, 4-color FACSCalibur (BectonDickinson) with antibody panels previously described[13 (link), 17 (link)]. For the remaining 63 patients, flow cytometry was performed with expanded flow protocols on the 3 laser, 10-color Gallios Flow Cytometer (Beckman Coulter). All 10-color procedures, antibodies, flow protocols, instrument settings, and gating strategies have been previously described by Gustafson et al[14 (link)]. Some patients were additionally stained with a modified T cell signaling protocol[18 (link)] to assess CD28 expression on CD3⁺CD56⁺ T cells. Analysis of flow data was performed using Cellquest (BD) or Kaluza (Beckman Coulter) software.