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Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry
Published on: January 7, 2019
Quantitative analysis of integrin expression in effusions using flow cytometric immunophenotyping
Eva Sigstad1, Hiep Phuc Dong, Søren Nielsen
1Department of Pathology (Section of Cytology), the Norwegian Radium Hospital, Oslo, Norway.
Insights
Flow cytometry (FCM) quantifies adhesion molecules in effusions, aiding diagnosis. High alpha6 integrin expression in malignant mesothelioma suggests laminin attachment, while alphaV/beta1 subunits indicate fibronectin/vitronectin binding.
Area of Science:
- Oncology
- Cell Biology
- Immunology
Background:
- Flow cytometric (FCM) immunophenotyping aids serous effusion diagnosis.
- Quantitative analysis of adhesion molecules in effusions requires further investigation.
Purpose of the Study:
- To evaluate flow cytometry (FCM) for quantitative analysis of adhesion molecule expression in serous effusions.
- To correlate adhesion molecule expression with specific effusion diagnoses (carcinoma, reactive, malignant mesothelioma).
Main Methods:
- Quantitative analysis of alphaV, alpha6, beta1, and beta3 integrin subunits using FCM on 67 effusion specimens.
- Utilized antibodies for epithelial (Ber-EP4, EMA), lymphoid (CD45), and mesothelial cell markers.
- Specimens were pre-diagnosed via morphology and immunocytochemistry.
Main Results:
- AlphaV, alpha6, and beta1 integrin subunits were frequently expressed across all groups.
- Significantly higher alpha6 subunit expression was observed in malignant mesothelioma (MM) (P = 0.029).
- Ber-EP4 and CD15 expression were significantly higher in carcinomas (P < 0.001, P = 0.001). EMA expression was higher in carcinomas and MM (P < 0.001).
Conclusions:
- FCM is an effective tool for quantitative adhesion molecule analysis in effusions.
- Elevated alpha6 integrin in MM suggests a role in laminin attachment.
- AlphaV and beta1 subunits indicate major extracellular matrix ligand binding in body cavities.
Abstract:
We have previously shown that flow cytometric (FCM) immunophenotyping is a useful adjunct to morphology, in the diagnosis of serous effusions. The objective of the present study was to evaluate the possible application of FCM to quantitative analysis of adhesion molecule expression in this clinical setting. Fresh frozen cells from 67 effusions underwent quantitative analysis of alphaV, alpha6, beta1, and beta3 integrin subunit expression, using FCM. Specimens were diagnosed as carcinoma (n = 48), reactive (n = 12), or malignant mesothelioma (MM; n = 7) using morphology and, in selected cases, immunocytochemistry prior to FCM analysis. Antibodies against established epithelial, lymphoid, and mesothelial cell epitopes (Ber-EP4, anti-epithelial membrane antigen; (EMA), anti-CD45, anti-CD14, and anti-CD15) completed the panel. Results (percentage of cells expressing the antigen) were analyzed for relationship with the morphologic diagnosis. Frequent expression of the alphaV, alpha6, and beta1 subunits was seen in all diagnostic groups, with significantly higher expression of the alpha6 subunit in MM (P = 0.029, Kruskal-Wallis H test). The beta3 integrin subunit was not detected in any of the specimens. Ber-EP4 and CD15 expression was significantly higher in carcinomas compared with reactive effusions and MM (P < 0.001 and P = 0.001, Kruskal-Wallis H test), and EMA expression was higher in carcinomas and MM, compared with reactive specimens (P < 0.001, Kruskal-Wallis H test). In conclusion, FCM is an efficient tool for quantitative analysis of adhesion molecules in effusions. The high alpha6 integrin subunit expression in MM suggests involvement of this receptor in tumor attachment to laminin. The frequent expression of the alphaV and beta1 subunits support attachment to fibronectin and vitronectin as the major ECM ligands in body cavities.
