title: Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: proposed practical method.
abstract: BACKGROUND
  Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell-based fluorescence assays performed on flow cytometers are currently lacking.
  
  
  METHODS
  Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined.
  
  
  RESULTS
  For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data-driven means for determining optimal replicate numbers.
  
  
  CONCLUSIONS
  The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes.
authors.affiliation: Trillium Diagnostics, LLC, Bangor, Maine, 04401.
authors.forename: Bruce H
authors.initials: BH
authors.lastname: Davis
authors.forename: Christine E
authors.initials: CE
authors.lastname: McLaren
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Carcio
authors.forename: Linda
authors.initials: L
authors.lastname: Wong
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Hedley
authors.forename: Mike
authors.initials: M
authors.lastname: Keeney
authors.forename: Adam
authors.initials: A
authors.lastname: Curtis
authors.forename: Naomi B
authors.initials: NB
authors.lastname: Culp
mesh_terms: D018952:Antigens, CD34 / Q000302:isolation & purification*; D005260:Female; D005312:Fetal Blood; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000473:pathology; D006801:Humans; D011247:Pregnancy; D018805:Sepsis / Q000097:blood*
chemical_list: D018952:Antigens, CD34; D005456:Fluorescent Dyes
keywords: IVD clearance; assay validation; fetomaternal hemorrhage test; flow cytometry; hENT1 assay; laboratory-developed test; quality assessment; regulatory science; sepsis test; stem cell assay