Figure 7.

Workflow illustrating the use of LCM coupled with RPPA in the diagnostic setting. First, tumor content in surgical samples and core needle biopsies is assessed by a certified pathologist and malignant cells are isolated from the surrounding microenvironment using LCM (A). Isolated cells are then lysed and immobilized onto nitrocellulose coated glass slides using a robotic system. Reference standard spanning the dynamic range of the analyte of interest and a set of internal controls are printed alongside with the clinical samples. Arrays are stained using an antibody-based detection system and absolute intensity values are generated for each sample and control (B). Intensity values of individual samples and controls are then interpolated from the reference standard and compared with a reference population matching the clinical characteristics of the samples (C). Expression levels of the measured analysis in the control samples are used as QA/QC steps to track precision and accuracy of the assay (C). Final results and QA/QC data are reviewed by a certified pathologist or a laboratory director and included in a final report (D). LCM, laser capture microdissection; PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; RPPA, reverse phase protein microarray.