title: Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot.
abstract: The ELISpot assay is used in vaccine studies for the quantification of antigen-specific memory B cells (B(MEM)), and can be performed using cryopreserved samples. The effects of cryopreservation on B(MEM) detection and the consistency of cultured ELISpot assays when performed by different operators or laboratories are unknown. In this study, blood was taken from healthy volunteers, and a cultured ELISpot assay was used to count B(MEM) specific for 2 routine vaccine antigens (diphtheria and tetanus toxoid). Results were assessed for intra- and inter-operator variation, and the effects of cryopreservation. Cryopreserved samples were shipped to a second laboratory in order to assess inter-laboratory variation. B(MEM) frequencies were very strongly correlated when comparing fresh and frozen samples processed by the same operator, and were also very strongly correlated when comparing 2 operators in the same laboratory. Results were slightly less consistent when samples were processed in different laboratories but correlation between the 2 measurements was still very strong. Although cell viability was reduced in some cryopreserved samples due to higher temperatures during transportation, B(MEM) could still be quantified. These results demonstrate the reproducibility of the ELISpot assay across operators and laboratories, and support the use of cryopreserved samples in future B(MEM) studies.
authors.affiliation: a Oxford Vaccine Group; Department of Paediatrics; University of Oxford and the NIHR Oxford Biomedical Research Centre; Oxford, UK.
authors.forename: Johannes
authors.initials: J
authors.lastname: Trück
authors.forename: Ruth
authors.initials: R
authors.lastname: Mitchell
authors.forename: Amber J
authors.initials: AJ
authors.lastname: Thompson
authors.forename: Begonia
authors.initials: B
authors.lastname: Morales-Aza
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Clutterbuck
authors.forename: Dominic F
authors.initials: DF
authors.lastname: Kelly
authors.forename: Adam
authors.initials: A
authors.lastname: Finn
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Pollard
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D015925:Cryopreservation / Q000379:methods*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D064368:Healthy Volunteers; D006801:Humans; D007156:Immunologic Memory; D007958:Leukocyte Count / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D011446:Prospective Studies; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*
chemical_list: D000942:Antigens, Bacterial
keywords: B cells; ELISpot; PBMC; cryopreservation; memory B cells; shipment; storage; vaccination