title: Stable Phenotype Of B-Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank.
abstract: 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 phenotypic differences between cryopreserved and fresh B-cell subsets". Subsequently, we performed a consecutive 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 Clinical Cytometry Society.
authors.affiliation: Department of Haematology, Aalborg University Hospital, Aalborg, Denmark.
authors.forename: Simon Mylius
authors.initials: SM
authors.lastname: Rasmussen
authors.forename: Anders Ellern
authors.initials: AE
authors.lastname: Bilgrau
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmitz
authors.forename: Steffen
authors.initials: S
authors.lastname: Falgreen
authors.forename: Kim Steve
authors.initials: KS
authors.lastname: Bergkvist
authors.forename: Anette Mai
authors.initials: AM
authors.lastname: Tramm
authors.forename: John
authors.initials: J
authors.lastname: Baech
authors.forename: Chris Ladefoged
authors.initials: CL
authors.lastname: Jacobsen
authors.forename: Michael
authors.initials: M
authors.lastname: Gaihede
authors.forename: Malene Krag
authors.initials: MK
authors.lastname: Kjeldsen
authors.forename: Julie Støve
authors.initials: JS
authors.lastname: Bødker
authors.forename: Karen
authors.initials: K
authors.lastname: Dybkaer
authors.forename: Martin
authors.initials: M
authors.lastname: Bøgsted
authors.forename: Hans Erik
authors.initials: HE
authors.lastname: Johnsen
keywords: B-cell subsets; Cryopreservation; Phenotyping