Kinetics of human myeloid-derived suppressor cells after blood draw

Abstract

BackgroundHuman myeloid-derived suppressor cells (MDSC) have been described as a group of immature myeloid cells which exert immunosuppressive action by inhibiting function of T lymphocytes. While there is a huge scientific interest to study these cells in multiple human diseases, the methodological approach varies substantially between published studies. This is problematic as human MDSC seem to be a sensible cell type concerning not only cryopreservation but also time point after blood draw. To date data on delayed blood processing influencing cell numbers and phenotype is missing. We therefore evaluated the kinetics of granulocytic MDSC (gMDSC) and monocytic MDSC (mMDSC) frequencies after blood draw in order to determine the best time point for analysis of this recently defined cell type.MethodsIn this study, we isolated peripheral blood mononuclear cells (PBMC) of patients with HIV infection or solid tumors directly after blood draw. We then analyzed the frequencies of gMDSC and mMDSC 2, 4 and 6 h after blood draw and after an overnight rest by FACS analysis using the standard phenotypic markers. In addition, part of the cells was frozen directly after PBMC preparation and was measured after thawing.ResultsgMDSC levels showed no significant difference using fresh PBMC over time with a limitation for the overnight sample. However they were massively diminished after freezing (p = 0.0001 for all subjects). In contrast, frequencies of fresh mMDSC varied over time with no difference between time point 2 and 4 h but a significantly reduction after 6 h and overnight rest (p = 0.0005 and p = 0.005 respectively). Freezing of PBMC decreased the yield of mMDSC reaching statistical significance (p = 0.04). For both MDSC subgroups, FACS analysis became more difficult over time due to less sharp divisions between populations.ConclusionsAccording to our data human MDSC need to be studied on fresh PBMC. gMDSC can be studied with delay, mMDSC however should be studied no later than 4 h after blood draw. These results are crucial as an increasing number of clinical trials aim at analyzing MDSC nowadays and the logistics of blood processing implies delayed sample processing in some cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0755-y) contains supplementary material, which is available to authorized users.

Clinical Significance of Circulating CD33+CD11b+HLA-DR− Myeloid Cells in Patients with Stage IV Melanoma Treated with Ipilimumab

“…We found that in contrast to blood samples separated by Ficoll density gradient (fresh or cryopreserved/thawed), only cryopreservation/thawing of the whole-blood sample preserves the levels of CD33þCD11bþHLA-DR À MDSCs as detected in fresh samples. These results are supported by previous studies showing that fresh or cryopreserved/thawed PBMCs, isolated on Ficoll density gradient, showed significantly modified frequencies of both CD33 þ CD11b þ HLA-DR À and CD15 þ LIN À HLA-DR À MDSC populations as compared with their profile in the whole fresh blood sample (22,23). It is important to note that in the process of the Ficoll density gradient separation step, due to the distribution of the different leukocyte populations, cells such as granulocytic MDSCs are neglected (missed) and only PBMCs containing the monocytic MDSCs are taken.…”

“…1C-D). These results are supported by previous studies showing that PBMC isolation using Ficoll before and after freezing, significantly changes the frequencies of both CD33 þ CD11b þ HLA-DR À and CD15 þ LIN À HLA-DR À MDSC populations after sample are thawed (22,23).…”

Myeloid cells in circulation and tumor microenvironment of breast cancer patients

“…Several studies have emphasized on the significance of sample handling and processing when monitoring MDSC levels in circulation and are in agreement over the adverse effects of cryopreservation and the delayed time points at which MDSC analysis was carried out following blood collection [20–22]. Mandruzzato et al suggested to perform analysis on fresh blood to prevent possible loss of some MDSC subsets mainly G-MDSC.…”

Endoplasmic reticulum stress induced LOX‐1+ CD15+ polymorphonuclear myeloid‐derived suppressor cells in hepatocellular carcinoma

“…One of the critical reasons is the heterogeneity of MDSC testing among different centres . At present, the standard method to separate MDSC is by density gradient centrifugation only in peripheral blood (PB) . This testing technology is not easily standardized between laboratories, which might be a critical cause of the heterogeneity of results in MDSC‐targeted studies.…”