Location, location, location: cytokine concentrations are dependent on blood sampling site

Abstract

Objective: Considerable breakthroughs in the field of sepsis have been made using animal models. Sepsis exhibits a wide array of derangements that may be evaluated in the blood, including the release of proinflammatory and anti-inflammatory cytokines. The Shock journal adheres to the ARRIVE guidelines regarding reporting in vivo results to allow reproducibility of data findings. It is generally assumed that blood cytokine concentrations collected from typical sampling sites will be similar, but there are no data validating that this is true. The main purpose of the present study was to determine if the location of blood sampling results in cytokine concentration differences following inflammatory insults.

Methods: Two different models of acute inflammation were studied. Adult, female ICR (Institute of Cancer Research) mice were injected with Escherichia coli lipopolysaccharide (n = 28) or subjected to cecal ligation and puncture (n = 16). They were killed at early time points following these inflammatory challenges for the collection of blood from the facial vein, retro-orbital sinus, and heart. Additional samples were collected in EDTA and heparin. Plasma cytokines from the same mouse were collected from each sampling site and evaluated by enzyme-linked immunosorbent assay. Clinical chemical parameters including plasma blood urea nitrogen and total protein were also analyzed.

Results: Regardless of model, time of collection, or cytokine measured, cytokine values from heart blood were higher than facial vein values from the same mouse. Interleukin (IL-6) collected from the heart relative to the facial vein demonstrated elevated concentrations following injection of lipopolysaccharide. In a similar manner, higher concentrations of IL-6, macrophage inflammatory protein 2, IL-10, and IL-1 receptor antagonist were found in cardiac puncture samples compared with other sampling sites 24 h after sepsis induced by cecal ligation and puncture. Similar differences were not seen when comparing blood urea nitrogen and total protein values from the two different sites. Using plasma IL-6 collected from the heart would incorrectly stratify predicted-to-live mice into the predicted-to-die category. Therefore, a simple linear regression model was developed to correctly restratify mice to their predicted fate. These data demonstrate that proinflammatory and anti-inflammatory cytokine concentrations are dramatically elevated when drawn centrally from the heart compared with collection from peripheral locations such as the facial vein. It is critical for publications to document the sampling location when evaluating plasma cytokines and attempting to compare studies.

FIGURE 1

Elevated plasma IL-6 after endotoxin injection when sampled by cardiac puncture relative to facial vein. (A) Mice (n = 8) underwent blood sampling from facial vein followed by cardiac puncture two hours following LPS, while (B) a separate group of mice (n = 10) underwent blood sampling from the same two sites 6 hours following injection. Plasma IL-6 collected by cardiac puncture was significantly elevated compared to IL-6 collected by facial vein in the same mouse. ** = p < 0.001 and *** = p < 0.0005.

Figure 2

Effect of anticoagulants on cytokine concentrations. Mice were injected with LPS and facial vein and cardiac blood collected two hours later in either EDTA or heparin. The anticoagulant did not alter IL-6 concentrations. However, cardiac values were still higher than facial vein samples. ** = p<.01 compared to cardiac samples for the same anticoagulant. N= 8-10 mice for each group.

FIGURE 3

IL-6 concentrations were similar when comparing plasma collected from facial vein and retro-orbital sinus, both of which are significantly diminished compared to cardiac puncture sampling. Blood was drawn 24 hours post CLP from common sites published in literature, including cardiac puncture, facial vein, and retro-orbital sinus in a subset of mice (n = 4). Plasma IL-6 collected by facial vein and retro-orbital sinus were calculated as a percent of IL-6 collected by cardiac puncture. It is clear that cytokine values following CLP collected by facial vein and retro-orbital sinus sampling were comparable to each other; while cytokines collected by cardiac puncture were elevated compared to other sampling sites.

FIGURE 4

Clinical parameters of other plasma measurements are similar regardless of sampling site. (A) Blood urea nitrogen (BUN) concentrations were measured in plasma collected 24 hours following CLP from the same group of mice that underwent CLP (n = 16). (B) There was no difference in total protein when comparing total protein collected from the two different sites. Unlike the differences seen in cytokine values from two different sampling sites, there was no significant difference in BUN or total protein levels in the samples collected by facial vein or cardiac puncture.

FIGURE 5

Misclassification of mortality prediction based on plasma IL-6. (A) All mice subjected to CLP were classified into Live-P, indeterminate, and Die-P based on specific plasma IL-6 values obtained by facial vein. (B) Mice stratified into the three predicted fates based on facial vein plasma IL-6 scale. Appreciating the scale on the y-axis would be vastly different and would result in misclassification if the much lower plasma cut off values used for the facial vein collected plasma were to be used. (C) A simple linear regression model was developed to help reclassify mice into their appropriate category for a more accurate prediction of mortality in acute phase of sepsis following CLP.