Validation of multiplex immunofluorescence and digital image analysis for programmed death-ligand 1 expression and immune cell assessment in non-small cell lung cancer: comparison with conventional immunohistochemistry

J Clin Pathol. 2022 Jul;75(7):452-458. doi: 10.1136/jclinpath-2021-207448. Epub 2021 Mar 29.


Aims: This study aimed to validate the application of combined multiplex immunofluorescence (mIF) and digital image analysis (DIA) in formalin-fixed and paraffin-embedded tissues for the quantitative assessment of programmed death-ligand 1(PD-L1) and immune cells (ICs) in non-small cell lung cancer (NSCLC).

Methods: Fifty resected samples of NSCLC were sequentially stained with a DNA-tagged mIF (panel including PD-L1, CKpan, CD8, CD68 and 4',6-diamidino-2-phenylindole (DAPI)) and conventional immunohistochemistry (cIHC). The assessment of cell density and consistency of tumour proportion score (TPS) via DIA were compared with those by pathologists.

Results: A strong correlation in the cell population of immune markers was obtained between mIF and cIHC (for PD-L1: R=0.9304, CKpan: R=0.8231, CD8: R=0.9314 and CD68: R=0.8366) within 95% limits of agreement. The continuous TPS calculated using mIF was highly consistent with the IHC staining results which were evaluated by pathologists (R=0.9362). However, in the comparison of TPS using interval variables, a poor agreement was obtained at a cut-off of 1% (κ=0.197), whereas excellent agreement was achieved at cut-offs of 50% (κ=0.908) and 5% (κ=0.823). DIA on mIF showed that PD-L1 commonly colocalised with CD68+ macrophages and CD8+ cytotoxic cells were closer to PD-L1-/CK+ tumour cells (TCs) than to PD-L1+/CK+ TCs in spatial distribution.

Conclusions: A combination of mIF and DIA is useful for the quantification of PD-L1 expression and IC populations in NSCLC. Further validation of TPS at a cut-off of 1% and assay harmonisation is essential for translating this method in a diagnostic setting.

Keywords: biomarkers; immunohistochemistry; lung neoplasms; tumor.

MeSH terms

  • B7-H1 Antigen / metabolism
  • Biomarkers, Tumor / analysis
  • Carcinoma, Non-Small-Cell Lung* / pathology
  • Fluorescent Antibody Technique
  • Humans
  • Immunohistochemistry
  • Lung Neoplasms* / pathology
  • Staining and Labeling


  • B7-H1 Antigen
  • Biomarkers, Tumor
  • CD274 protein, human