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Stable phenotype of B ‐cell subsets following cryopreservation and thawing of normal human lymphocytes stored in a tissue biobank
Author(s) -
Rasmussen Simon Mylius,
Bilgrau Anders Ellern,
Schmitz Alexander,
Falgreen Steffen,
Bergkvist Kim Steve,
Tramm Anette Mai,
Bæch John,
Jacobsen Chris Ladefoged,
Gaihede Michael,
Kjeldsen Malene Krag,
Bødker Julie Støve,
Dybkær Karen,
Bøgsted Martin,
Johnsen Hans Erik
Publication year - 2015
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21192
Subject(s) - cryopreservation , biology , flow cytometry , phenotype , andrology , cd19 , cd146 , immunology , microbiology and biotechnology , gene , genetics , cd34 , embryo , medicine , stem cell
Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B‐cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that “overall, there are no phenotypic differences between cryopreserved and fresh B‐cell subsets.” Subsequently, we performed an uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3 , respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B‐cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B‐cell hierarchy; however, storage procedures need to be delineated by tissue‐specific comparative analysis. © 2014 International Clinical Cytometry Society