title: Correcting the Relative Bias of Light Obscuration and Flow Imaging Particle Counters.
abstract: OBJECTIVE
  Industry and regulatory bodies desire more accurate methods for counting and characterizing particles. Measurements of proteinaceous-particle concentrations by light obscuration and flow imaging can differ by factors of ten or more.
  
  
  METHODS
  We propose methods to correct the diameters reported by light obscuration and flow imaging instruments. For light obscuration, diameters were rescaled based on characterization of the refractive index of typical particles and a light scattering model for the extinction efficiency factor. The light obscuration models are applicable for either homogeneous materials (e.g., silicone oil) or for chemically homogeneous, but spatially non-uniform aggregates (e.g., protein aggregates). For flow imaging, the method relied on calibration of the instrument with silica beads suspended in water-glycerol mixtures.
  
  
  RESULTS
  These methods were applied to a silicone-oil droplet suspension and four particle suspensions containing particles produced from heat stressed and agitated human serum albumin, agitated polyclonal immunoglobulin, and abraded ethylene tetrafluoroethylene polymer. All suspensions were measured by two flow imaging and one light obscuration apparatus. Prior to correction, results from the three instruments disagreed by a factor ranging from 3.1 to 48 in particle concentration over the size range from 2 to 20 μm. Bias corrections reduced the disagreement from an average factor of 14 down to an average factor of 1.5.
  
  
  CONCLUSIONS
  The methods presented show promise in reducing the relative bias between light obscuration and flow imaging.
authors.affiliation: Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA. dean.ripple@nist.gov.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Ripple
authors.affiliation: Center for Computational and Systems Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Zhishang
authors.initials: Z
authors.lastname: Hu
mesh_terms: D003952:Diagnostic Imaging / Q000379:methods*; D005466:Fluorocarbons / Q000737:chemistry; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry; D008027:Light; D010316:Particle Size; D011108:Polymers / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D012827:Silicone Oils / Q000737:chemistry; D013535:Suspensions / Q000737:chemistry*; D011138:Polytetrafluoroethylene / Q000031:analogs & derivatives
chemical_list: D005466:Fluorocarbons; D007136:Immunoglobulins; D011108:Polymers; D012709:Serum Albumin; D012827:Silicone Oils; D013535:Suspensions; C040378:poly(ethylene-tetrafluoroethylene); D011138:Polytetrafluoroethylene
keywords: flow imaging; flow microscopy; light obscuration; particle; protein aggregate