title: Evaluation of peripheral blood mononuclear cell processing and analysis for Survival Motor Neuron protein.
abstract: OBJECTIVE
  Survival Motor Neuron (SMN) protein levels may become key pharmacodynamic (PD) markers in spinal muscular atrophy (SMA) clinical trials. SMN protein in peripheral blood mononuclear cells (PBMCs) can be quantified for trials using an enzyme-linked immunosorbent assay (ELISA). We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal.
  
  
  METHODS
  Several variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers.
  
  
  RESULTS
  Delaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability (>90%). SMN levels from PBMCs isolated by EDTA tubes/Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3-5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age.
  
  
  CONCLUSIONS
  The sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.
authors.affiliation: Spinal Muscular Atrophy Foundation, New York, New York, United States of America. dkobayashi@smafoundation.org
authors.forename: Dione T
authors.initials: DT
authors.lastname: Kobayashi
authors.forename: Douglas
authors.initials: D
authors.lastname: Decker
authors.forename: Phillip
authors.initials: P
authors.lastname: Zaworski
authors.forename: Karen
authors.initials: K
authors.lastname: Klott
authors.forename: Julie
authors.initials: J
authors.lastname: McGonigal
authors.forename: Nabil
authors.initials: N
authors.lastname: Ghazal
authors.forename: Laurel
authors.initials: L
authors.lastname: Sly
authors.forename: Brett
authors.initials: B
authors.lastname: Chung
authors.forename: James
authors.initials: J
authors.lastname: Vanderlugt
authors.forename: Karen S
authors.initials: KS
authors.lastname: Chen
mesh_terms: D000293:Adolescent; D000328:Adult; D002353:Carrier State / Q000097:blood; D002452:Cell Count; D002469:Cell Separation; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007223:Infant; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009134:Muscular Atrophy, Spinal / Q000097:blood; D012141:Respiratory Tract Infections / Q000097:blood; D055532:SMN Complex Proteins / Q000378:metabolism*; D055815:Young Adult
chemical_list: D055532:SMN Complex Proteins