Many studies have reported gene expression differences in PBMCs without providing details of sample processing procedures. This study highlights the importance of considering processing delays since periods as short as 4
h can have significant effects on gene expression. While additional studies with independent samples are necessary to define specific processing gene expression signatures, it is clear that processing delays must be considered in experimental design and data interpretation.
This study was not designed to determine the specific cause of the identified gene expression changes although several explanations can be hypothesized. Phlebotomy itself can have an influence on lab results obtained from blood samples.10
Regarding storage of samples after phlebotomy, a previous study looked at the effect of room temperature storage of serum and plasma.11
h after phlebotomy, the environment of the plasma and serum sample changed significantly, which would cause biological stresses. Glucose levels dropped, lactate increased, pH decreased, and hypoxia were identified by a decrease in pO2
and an increase in pCO2
. Although this study did not examine the effect of blood processing delays, it can be expected that similar effects would be encountered. Even the simple preanalytical variables of the additive in the collection tube (eg, ACD in this study) or interaction of cells with the surface of the tube itself (a significant change from the vasculature of the donor) for several hours may cause the identified gene expression changes.
Proteins derived from the genes listed in are important in inflammatory, autoimmune, and cancer pathways. Vascular endothelial growth factor (VEGF) is a prototypical angiogenic factor,12
and anti-VEGF has become the standard treatment for many tumor types.13
CCR2, CCR5, and IL-8 are chemoattractants that activate and recruit immune cells to sites of infection and inflammation. Antagonists to CCR2 have been suggested as therapy for a variety of inflammatory diseases as well as obesity and pulmonary disease.14
The CCR5 (also called CD195) antagonist, Maraviroc, is currently approved for treatment of HIV infection.15
SOCS2 and 3 are key negative regulators of cytokine signaling.16
TLR10 and CD180 (RP105) are involved in toll-like receptor signaling. The fact that these genes have variable expression attributable to processing delays emphasizes the importance of attention to preanalytic variables in sample collection and processing, a fundamental component of biospecimen science.
Various strategies can be employed to address possible changes caused by processing delays. The method we chose to reduce the impact of this variable was to remove samples with extended processing times (>4
h) from analysis.6–8
As an alternative, time-sensitive genes may be removed from consideration.17
Both of these methods have the benefit of simplicity although specific cutoffs are arbitrary. A disadvantage of this approach is that time-sensitive genes might also be involved in pathogenic processes. An interesting, but untested, method would be to delay processing of all samples until a specified time postphlebotomy (eg, 2
h) to reduce this variability. Alternatively, statistical approaches such as linear modeling may be employed using processing time as 1 variable.
To avoid the effect of variable processing time, technologies that stabilize samples instantly have been developed. These methods collect whole blood, including neutrophils that tend to vary in number more than other cellular components, and metabolically active immature red blood cells. Together, these components may overshadow more biologically relevant PBMCs. Additionally, there are issues with the so-called globin effect, where excessive amounts of globin mRNA derived from the reticulocytes present in whole blood interfere with the microarray analysis as seen by decreased present calls.5
It is unclear if this occurs due to inhibition during the labeling or probing steps of the assay. Newer labeling systems may help overcome this issue (as indicated by recovery of detected genes; unpublished data).
It is evident from this study that processing delays affect gene expression patterns obtained from PBMCs in a very short period. It is, therefore, important for this variable to be measured so that its effect can be considered in data interpretation.