Peripheral blood manipulation significantly affects the result of dendritic cell monitoring

It has been postulated that the plasmacytoid/myeloid dendritic cell ratio (pDC/mDC) reflects immune reactivity, and can therefore be used to monitor transplant recipients. We investigated the influence of Ficoll-Paque separation and PBMC cryopreservation on the pDC/mDC ratio and the expression of maturation markers, e.g. chemokine receptors (CKRs) CCR7, CXCR4, and CCR5, in comparison to fresh blood cells. Fractions of pDCs and mDCs, and CKR expression were measured by flow cytometry in fresh blood, in Ficoll-isolated PBMCs and in cryopreserved PBMCs from healthy individuals and kidney transplant recipients. Ficoll-isolation of PBMCs resulted in higher pDC/mDC ratios in both groups compared to fresh blood cells resulting from a relatively large increase in pDCs compared to mDCs. The pDC/mDC ratio increased further after cryopreservation of PBMCs from kidney transplant recipients. Ficoll-isolation and cryopreservation of PBMCs affected the proportion of mDCs and pDCs positive for CKRs, and their expression levels resulting in a more mature phenotype. In conclusion, the pDC/mDC ratio and pDC or mDC maturation status based on CKR expression, is dependent on manipulation of PBMCs. Therefore, fresh blood is preferable for monitoring purposes in transplant patients, as only these cells reflect the in vivo immune-status of patients accurately.