Granulocytic myeloid-derived suppressor cells (MDSC) are a MDSC subset expanded in various cancer types. As many clinical studies rely on the use of stored collections of frozen blood samples, we first tested the influence of freezing/thawing procedures on immunophenotyping and enumeration of granulocytic MDSC (G-MDSC). To identify factors involved in expansion of human G-MDSC, we then analyzed correlations between G-MDSC frequencies, clinical parameters and granulocyte-related factors in the peripheral blood of head and neck cancer patients. HLA-DR, CD14, CD33 and CD66b allowed a clear discrimination of G-MDSC from monocytic MDSC and immature myeloid cells. MDSC subsets were sensitive to cryopreservation with immature G-MDSC showing the highest sensitivity. G-MDSC frequencies were increased in advanced disease stage and associated with the level of CCL4 and CXCL8, but not with colony-stimulating factors, IL-6, S100A8/9, CXCL1 and other cytokines. Our results indicate that the frequency of MDSC, in particular G–MDSC, may be underestimated in retrospective clinical analyses using frozen blood samples. Increased G-MDSC frequencies correlate with advanced disease and increased concentrations of CXCL8, but, unexpectedly, not with growth factors (such as granulocyte colony-stimulating factor), IL-6 and CXCL1. Our data suggest that CXCL8 promotes accumulation of G-MDSC in cancer patients independent of classical colony-stimulating factors.