Harmonized and controlled experimental methods are critical for all clinical and non-clinical human studies. Routinely, the checkpoints for quality control of blood samples and its derivatives rest on variables such as sample processing procedures, anticoagulants used, temperatures, incubation/exposure times, brand and lot of reagents, among others. However, sample pre-processing parameters that could influence the quality and stability of the biological material such as time-to-sample processing or time of day for sample procurement are not often considered. Results from this study revealed that a time delay between obtaining a peripheral blood sample and its processing, affects inflammatory gene expression and potentially cell functions, providing critical evidence of the importance of controlling blood sample handling variables prior to ex-vivo analyses.
Previous studies have evaluated the impact of time after phlebotomy and its relationship with the quality of data derived from immunological analyses [12, 17,18,19,20,21]. Despite their important contribution, these studies evaluated the effect of sample processing delay in a single immunologic parameter (cell function, cell count, activation markers or gene expression), and usually during just one time point (most of them after a 24 h time frame), limiting the accurate definition of sample processing times, especially when studies involve multiple clinical sites and multiple immunological parameters. To overcome this, our approach aimed to concomitantly analyze the dynamic effects of time to blood sample processing on multiple immune and cellular parameters.
Although not statistically significant, our results showed a reduction in the cellular viability at 24 h, which could have important biological implications. Similar findings were previously reported, where average viability within 8 h of processing was ∼92%, decreasing significantly to ∼84% when processing was delayed 24 h, concluding that for optimal cell viability and recovery, PBMCs must be processed and cryopreserved within 8 h after venipuncture . Our data extend these observations and indicate that in terms of cell viability, blood samples could be processed up to 12 h after phlebotomy.
In the context of immunological assays and analyses of cell function, the time frame between phlebotomy and PBMCs isolation is critical [15, 18]. A delay in the processing of PBMCs over 8 h, has been shown to result in alterations of NK cells functions: decreased expression of chemokine receptors (CCR4 and CCR7), reduction of degranulation capacity and secretion of IFNγ and TNFα . Similarly, a progressive impairment of antigen presenting cell function of plasmacytoid and myeloid dendritic cells and monocytes has been reported at 0, 6, 12 and 24 h between venipuncture and PBMC isolation . Our data showed that the frequency of monocytes, NK cells, and B cells in whole blood was unaffected at early time points and up to 24 h after phlebotomy, concurring with prior studies and expanding towards earlier time-points of analysis . However, our results revealed a significant decrease in the CD3+ T cell population at 24. Loss of CD3+ T cells occurred concomitantly with increase in the CD3 negative lymphocyte population, suggesting antigen loss rather than an impact on cell viability. Rapid internalization and recycling of the TCR:CD3 complex has been reported in resting T cells , potentially explaining the observed reduction in the CD3+ T cell population.
In the context of immune-related gene expression, we observed different patterns of expression among the analyzed genes. Whereas expression of TNFα was strongly upregulated in a time-dependent manner, and CCL8, CCR2 and CXCL10 were down-regulated at all time points after 2 h, and the expression of CXCL3, CCL2, IL-1β and CCR7 remained unchanged. Expression profiling in PBMCs using microarrays from human blood samples processed immediately or the next day after overnight incubation revealed that 31.7% of genes (2034 of the 6414 evaluated genes) were sensitive to ex-vivo incubation. A high proportion of these genes were involved in basic cellular processes such as transcriptional regulation, cell cycle progression, TNF-related functions, immune signaling, apoptosis and cytokines/chemokines signaling . Interestingly, changes in gene expression have been reported to occur as early as 4 h post venipuncture . These processes may reflect transcriptional events as an active response to cellular stress, and/or effects induced by alterations in cell-to-cell contact dynamics, which alert to an understanding of the basal gene expression patterns analyzed in blood samples prior to functional interpretation of gene expression profiles.
This study explored the effect of time to blood sample processing on a spectrum of immunological readouts. Overall, our findings indicated that blood samples processed after 12 h of phlebotomy should be carefully controlled and critically analyzed as alterations in cell viability and functionality are evidenced. This has important implications in multi-center/multi-site studies and field studies where time control can be challenging. Potential strategies to overcome this could include definition of a single time-point for sample processing within all clinical sites, inclusion of time-specific control samples to be collected simultaneously to the study samples (e.g. a blood sample from a study subject processed in parallel to one of a healthy volunteer as a normalizing control). Results presented herein provide an evidence base for the design of studies that involve human blood sampling and downstream analyses of immunological functions.