Creating a robust framework for the analysis of cryopreserved samples in quantitative immunological experiments
Faculty of Medicine and Health Sciences
Journal of immunological methods. - Amsterdam
, p. 63-67
University of Antwerp
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.