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 time 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 secreted by lymphocytes across varying storage time by flow cytometry to study the changes in cytokine expression and 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 within the 7-day storage period. Moreover, prolonged storage time markedly reduced the phosphorylation of signal transducer and activator of transcription 4 (STAT4) in lymphocytes, which was accompanied with decreased IFN-γ secretion. Our results indicated that failure to perform timely testing would lead to inappropriate understanding of cell quantity and state in clinical practice. This study provides a reference for clinical choice of optimal sample testing time, thereby minimizing potential biases introduced by suboptimal storage time.