Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot

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.