The effect of storage time on lymphocytes function detection: Application in clinical samples

Due to the complexity of clinical work, some blood samples for lymphocyte activity detection might be conducted thrice weekly rather than daily. Whether the levels of cytokines secreted by lymphocytes are affected by storage days of blood samples has not been studied. In clinical work, we found that many samples that were not tested on the day of blood collection had lower levels of interferon secretion than normal. Consequently, we assessed cell counts and cytokines secretion by lymphocytes across varying storage durations by flow cytometry to study cytokine changes, to determine optimal sample processing timing. We found that with the extension of storage time, the proportion and absolute count of various lymphocytes were significantly decreasing. Particularly, natural killer cells (NK cells), CD4+ T cells, CD8+ T cells and NKT-like cells all showed a decreased interferon γ (IFN-γ) secretion as early as the second day of blood collection. The levels of TNF-α secreted by CD8+ T cells and NKT-like cells also decreased with storage time. The expression of granzyme B and perforin by various lymphocytes did not change with the 7 days of storage. Moreover, the phosphorylation of signal transducer and activator of transcription 4 (STAT4) was significantly reduced in lymphocytes with storage time. Our results indicated that failure to perform timely testing would lead to inappropriate understanding of cell quantity and state in clinical practice. Our research provides a reference for clinical selection of sample testing time to avoid the inaccuracy of results due to long-term storage time.