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Updated: Aug 8, 2026
A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes
Published on: December 6, 2014
Sampling tumor-draining lymph nodes for phenotypic and functional analysis of dendritic cells and T cells
Ronald J C L M Vuylsteke1, Paul A M van Leeuwen, Sybren Meijer
1Department of Surgical Oncology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands.
Insights
Scraping lymph nodes (LNs) offers a viable alternative to tissue dissociation for isolating immune cells. This method effectively preserves cell viability and function for immune response analysis in sentinel nodes (SNs).
Area of Science:
- Immunology
- Oncology
- Surgical Pathology
Background:
- Immune responses to tumors initiate in sentinel lymph nodes (SNs).
- Conventional methods for isolating immune cells from SNs can be hindered by diagnostic procedures, impacting functional analysis.
- There is a need for alternative methods to obtain viable immune cells from lymph nodes (LNs).
Purpose of the Study:
- To investigate and compare the efficacy of LN scraping versus traditional LN dissociation for isolating viable immune cells.
- To assess the phenotypic and functional characteristics of immune cells obtained through both methods.
- To determine if LN scraping is a feasible alternative for analyzing immune responses in SNs.
Main Methods:
- Tumor-draining lymph nodes (LNs) were obtained from five cancer patients.
- Immune cells were isolated using two methods: scraping LNs with a surgical blade and dissociating total LNs.
- Phenotypic characterization was performed using flow cytometry.
- Functional analysis included antigen-specific interferon-gamma (IFN-γ) release assays (ELISPOT).
Main Results:
- Both scraping and dissociation methods yielded comparable cell viabilities and phenotypic characteristics.
- T-cell functionality, including expansion factors and cytotoxic T-cell frequencies, was similar between the two isolation methods.
- Specific T-cell recognition of influenza or Her-2/neu tumor antigens was comparable.
Conclusions:
- LN scraping is a feasible and effective method for obtaining viable immune cells for functional analysis.
- This technique serves as a valuable alternative to LN dissociation, especially for small LNs designated for histological examination.
- Scraping LNs can aid in monitoring immune responses within sentinel nodes (SNs).
Abstract:
Immune responses against tumor antigens will initially occur in the first tumor-draining lymph node, the sentinel node (SN). Because of extensive diagnostic procedures, obtaining a piece of SN to isolate viable immune cells for functional analyses is often impossible. For this reason an alternative method to obtain viable cells from a lymph node (LN) was investigated, ie, scraping LNs with a surgical blade, and compared with dissociation of total LNs. Tumor-draining lymph nodes were retrieved from five oncological patients. The collected dendritic cells and T cells were phenotypically and functionally characterized by flow cytometry and antigen-specific interferon (IFN)-gamma release in an ELISPOT assay. Results were compared between the two isolation methods. Viabilities and phenotypic characteristics of the collected cells were entirely comparable for both methods. T-cell functionality was also comparable between both methods, with equal T-cell expansion factors and similar frequencies of cytotoxic T cells specifically recognizing the M1 matrix protein of Influenza haemophilus or the tumor antigen Her-2/neu. In conclusion, scraping LNs to obtain cells for analysis of immune functions in LNs is feasible and presents a good alternative to dissociation of LNs. Scraping may even be applied to small LNs that a pathologist will submit entirely for histological examination and may thus prove useful in the monitoring of immune responses in SNs.
