Creating a robust framework for the analysis of cryopreserved samples in quantitative immunological experiments.

Longitudinal clinical or experimental immunological studies warrant the use of cryopreserved samples for flow cytometric phenotyping. Most notably CD62L+ and CD25+Foxp3+ counts were shown to be reduced by past studies. Here we are the first to compare the effects of cryopreservation on cell type calculations performed on a longitudinal dataset. We first compared lymphocyte subpopulation counts from fresh samples with those from samples frozen for either 5 or 6 months coming from 9 individuals. This way, we found that the cell counts obtained after basic lymphocyte differentiation in CD3+CD4+, CD3+CD8+, CD3-CD19+ and CD3-CD56+ were relatively robust for cryopreservation. However, when further subtyping CD4+ and CD8+ cells, we only found CCR7 and CD45RA to have a relation between fresh and cryopreserved counts, but we could not conclude the same for CD62L. Also, CD4+CD25+Foxp3+ were shown to be approximately 0.5 times less counted after cryopreservation. Next, we performed basic longitudinal calculations for which we either subtracted the cell counts at time 1 from the cell counts at time 2 or calculated the ratio between the cell counts at time 2 and time 1, for both fresh and cryopreserved samples. This way, we found that the use of absolute cell counts supported a good one-to-one relation between fresh and cryopreserved counts for all markers except CD62L and CD4+CD25+Foxp3+. In conclusion, we found no support for the use of CD62L and CD4+CD25+Foxp3+ as markers for calculations on flow cytometric counts from cryopreserved longitudinal datasets. However, all other basic lymphocyte markers proved to be relatively robust if absolute counts were used.