Feasibility of flowcytometric quantitation of immune effector cell subsets in the sentinel lymph node of the breast after cryopreservation

The sentinel lymph node (SLN) is an emerging focus for immunological research in breast cancer. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This is of particular importance for immunomonitor...

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Bibliographic Details
Published in:Journal of immunological methods Vol. 375; no. 1-2; pp. 189 - 195
Main Authors: van Pul, Kim M., Vuylsteke, Ronald J.C.L.M., Bril, Herman, Stockmann, Hein B.A.C., de Gruijl, Tanja D.
Format: Journal Article
Language:English
Published: Amsterdam Elsevier B.V 31.01.2012
Elsevier
Subjects:
Aged
Biological and medical sciences
Breast cancer
breast neoplasms
Breast Neoplasms - immunology
Breast Neoplasms - metabolism
Breast Neoplasms - pathology
CD4 antigen
CD4-CD8 Ratio - methods
CD8 antigen
CD8-Positive T-Lymphocytes - immunology
CD8-Positive T-Lymphocytes - metabolism
CD8-Positive T-Lymphocytes - pathology
CD83 antigen
CD86 antigen
Clinical trials
Cryopreservation
Cryopreservation - methods
Data processing
Dendritic cells
Dendritic Cells - immunology
Dendritic Cells - metabolism
Dendritic Cells - pathology
Effector cells
Female
Flow cytometry
Flow Cytometry - methods
Forkhead Transcription Factors - immunology
Forkhead Transcription Factors - metabolism
Foxp3 protein
freeze-thaw cycles
Freeze-thawing
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Humans
Immunophenotype
Lymph nodes
Lymph Nodes - immunology
Lymph Nodes - metabolism
Lymph Nodes - pathology
Lymphocytes T
Middle Aged
Molecular immunology
patients
Quantitation
quantitative analysis
Regulatory T cells
Sentinel lymph node
Sentinel Lymph Node Biopsy - methods
T-Lymphocyte Subsets - immunology
T-Lymphocyte Subsets - metabolism
T-lymphocytes
Techniques
thawing
variance
cDC
Dendritic cells
Immunophenotype
DMSO
Sentinel lymph node
CM
FACS
Quantitation
Regulatory T cells
MFI
SLN
Cryopreservation
FCS
DC
Dendritic cell
Lymph node
T-Lymphocyte
Cell subpopulation
Quantitative analysis
Immunological method
ISSN:0022-1759, 1872-7905, 1872-7905
Online Access:Get full text
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Summary:The sentinel lymph node (SLN) is an emerging focus for immunological research in breast cancer. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This is of particular importance for immunomonitoring of large multicenter trials. However, little data are available regarding the influence of cryopreservation on phenotypic characteristics of lymph node dendritic cells and T cells. In this study we assessed the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples. SLN were collected from nine breast cancer patients. From each SLN cell sample, half was used for immediate analysis and half was analyzed after cryopreservation and thawing. Conventional dendritic cell (cDC) and T cell subsets were quantified and phenotypically characterized by flow cytometry. The observed frequencies of both CD1a+ and CD1a−CD11c+CD14− cDC subsets showed significant correlation between the fresh and frozen-thawed samples. Similar high correlations were found for CD83 and CD86 expression markers on the more frequent (>0.2%) CD1a+ and CD1a−CD11c+CD14− cDC subset, but not on the low-frequency (<0.2%) CD1a+CD11c+CD14+ cDC subset. CD4/CD8 T cell ratios were comparable and were significantly correlated pre- and post-freezing. Regulatory CD4+CD25hi T cell frequencies and their FoxP3 expression levels were significantly higher after freezing–thawing than in the freshly analyzed samples. Nevertheless, a highly significant correlation was found for both parameters pre- and post-freezing. Cryopreservation and thawing seems a valid and practical alternative to direct analysis of fresh viable lymph node cells, without introducing cryo-dependent variance between SLN samples. However, enumeration of low-frequency cell populations and assessment of their marker expression levels are less reliable after cryopreservation and should be assessed and considered in the design of each clinical trial. ► Sentinel lymph node (SLN) is a popular focus for tumor immunological research. ► Influence of cryopreservation on phenotype of SLN cDC- and T-cells is not known. ► Flowcytometric analyses of fresh and cryopreserved SLN samples were compared. ► cDC and T cell frequencies correlated significantly pre- and post-cryopreservation. ► Cryopreservation is a valid alternative to direct analysis of fresh lymph node cells.
Bibliography:http://dx.doi.org/10.1016/j.jim.2011.10.011
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ISSN:0022-1759
1872-7905
1872-7905
DOI:10.1016/j.jim.2011.10.011