System Variability Study of Multiplexed Immunofluorescence Using Cell Line Microarrays

• Activity Number: 341
• Type: Contributed
• Date/Time: Tuesday, July 31, 2012 : 10:30 AM to 12:20 PM
• Sponsor: Biometrics Section
• Author(s): Colin C McCulloch*+ and Mike Lazare and Sireesha Kaanumalle and Natalia Jun and Rodney Beck and Kevin Kenny and Dave Henderson and Alex Corwin and Denise Hollman and Doug VanDerwerken
• Companies: GE Global Research and GE Global Research and GE Global Research and GE Healthcare and GE Healthcare and GE Global Research and GE Global Research and GE Global Research and GE Healthcare and Duke University
• Keywords: Random effects ; Gauge R&R ; Image analysis ; Biomarker

Abstract

Immunohistochemistry has traditionally been performed with an enzymatic system like diaminobenzidine (DAB) staining, which is limited by low dynamic range and number of proteins that can be probed for in a single tissue section. Multiplexed immunofluorescence (MI) is a potential solution to these limitations but first its sensitivity, repeatability, and reproducibility must be shown. A series of studies are described which demonstrate sensitivity and low technical variability of MI systems installed at three GE labs using cell-line microarrays stained for 4 proteins. Specification limits were defined on (1) concordance between automatic MI scores and manual DAB scores and (2) contribution of technical variability to total observed variability of the MI scores. Proportional odds, logistic regression, and hierarchical linear models were used. Performance was excellent at all three labs: MI / DAB concordance exceeded the specifications in all but a handful of the 56 slides processed, and technical variance was < 16% of total observed variance at all 3 labs. The main source of technical variability was poor cell adhesion to the slide which is a known limitation of cell line arrays.