Stimulated Whole Blood Cytokine Release as a Biomarker of Immunosuppression in the Critically Ill: The Need for a Standardized Methodology

ABSTRACT Objective: Reduced ex vivo lipopolysaccharide (LPS) stimulated whole blood pro-inflammatory cytokine release is a hallmark of immunosuppression in the critically ill and predicts adverse clinical outcomes. No standard technique for performing the assay currently exists. The impact of methodological heterogeneity was determined. Design, Setting, Subjects, and Interventions: Clinical experimental study set in a research laboratory. Venous blood from 5 to 10 healthy volunteers/experiment (total participant group: 18 subjects, 72% men, mean age 32) was stimulated ex vivo to evaluate the effect of variables identified via literature review on tumor necrosis factor-α (TNFα) release. These included sample handling, stimulation technique, and incubation conditions. Reporting convention was additionally assessed. Main Results: Measured TNFα release was significantly altered by source of LPS, concentration of LPS employed, duration and temperature of incubation prior to supernatant aspiration, and predilution of blood (repeated measures ANOVA, all P < 0.01). Sample handling prior to stimulation (anticoagulant employed, time to LPS addition, and storage temperature) also caused significant alterations in TNFα release. Considerable interindividual variation was observed (range 1,024–4,649 pg/mL, mean 2,339 pg/mL). Normalization by monocyte count and pretreatment with a cyclooxygenase inhibitor (indomethacin 10 μM) reduced the coefficient of variation from 47.17% to 32.09%. Conclusions: Inconsistency in interlaboratory methodology and reporting impairs interpretation, comparability, and reproducibility of the ex vivo LPS-stimulated whole blood cytokine release assay. A standardized validated technique is required. The advent of trials of immunoadjuvant agents renders this a clinical imperative.


INTRODUCTION
Late mortality in critically ill patients with severe sepsis, or following trauma, burn or major surgery, is now thought due to the predominance of anti-inflammatory processes. Whether excessive, sustained, or secondary to a failure of the immune system to recover from their functional consequences, the net effect is one of immunosuppression characterized by susceptibility to opportunistic infection and acquisition of secondary sepsis (1,2). Therapeutically, this may be addressed via augmentation of the immune system with immunoadjuvant agents (3), an approach that has demonstrated initial promise in selected septic patients (4,5).
The key to an immunorestorative strategy is patient stratification-identifying individuals with clinically meaningful immunosuppression who may benefit from treatment. To date, two interrelated biomarkers have repeatedly proven predictive of acquisition of both nosocomial infection and mortality and have consequently been used to guide inclusion in clinical trials: monocyte human leukocyte antigen DR (mHLA-DR) expression (6,7) and ex vivo lipopolysaccharide (LPS)-stimulated whole blood (WB) tumor necrosis factor a (TNFa) release (8)(9)(10). While a standardized methodology and reporting tradition has been established for mHLA-DR (11), the same is not true for ex vivo cytokine release, potentially leading to disparities in case definition, identification, treatment allocation, and outcome determination that impair data interpretation and comparability.
This study sought to identify key inter-laboratory differences in sample handling, technical performance of the WB ex vivo cytokine release assay, and result reporting convention, determining their impact on measured TNFa and hence quantification of immune competence. ). In addition, the effect of pretreating the blood with a non-steroidal anti-inflammatory drug (indomethacin 10 mM [Sigma-Aldrich], a dual cyclooxygenase 1 and 2 inhibitor) was evaluated. Where not supplied in solution LPS was reconstituted in sterile deionized water and vortexed extensively prior to use.

MATERIALS AND METHOD
TNFa and Monocyte Quantification: The concentration of TNFa was determined via enzyme-linked immunosorbent assay according to the manufacturer's instructions (Duoset, R&D systems, Minneapolis, MN). Differential leucocyte counts were performed via a Sysmex XE2100 flow cytometric analyzer by The Doctors Laboratory (London, UK).

Statistical analysis
Data points represent the average of three technical repeats, each assayed in duplicate. The D'Agostino-Pearson test was used to test for normality. Differences between categorical variables were tested for using either repeated measures one-way or two-way analysis of variance (RM-ANOVA, incorporating the Greenhouse-Geisser correction) with Tukey multiple comparisons test, or paired t tests. Statistical significance was set at P < 0.05. All analyses were conducted in Prism 6 (GraphPad Software).

RESULTS
Stimulation with LPS (1 ng/mL) from different bacterial sources elicited significantly different concentrations of TNFa (P < 0.001, one-way RM-ANOVA, Fig. 1A), with ''rough''-LPS derived from S. minnesota causing significantly greater TNFa release than the 3 ''smooth''-LPS sources (all P < 0.01, Tukey). Cytokine release was further altered by the use of alternate anticoagulants (P < 0.001, one-way RM-ANOVA) being significantly lower when EDTAwas employed than either LH or Na Cit (both P < 0.01, Tukey, Fig. 1C). While incubation of stimulated WB at 378C was necessary-samples at 208C releasing minimal TNFa (mean 89.27 pg/mL, Fig. 1E) -no significant difference was observed between agitated (mean 4,049 pg/mL, SD 1653) and nonagitated samples (4,459 pg/mL, SD 1967; P ¼ 0.15, paired t test). Stimulation with either increasing doses of LPS (S. abortus equi), or for variable periods of time, predictably led to differential concentrations of TNFa in the supernatant when aspirated; 1 ng/mL and incubation time >4 h leading to maximal or equivalent responses ( Fig. 1B and D). Spontaneous production of TNFa in un-stimulated blood was not observed.
Variations in sample handling prior to LPS stimulation caused discrepancies in assayed TNFa. Both time to LPS addition and storage temperature significantly effected supernatant TNFa concentration (both P < 0.001, 2-way RM ANOVA), increasing time leading to lower release and 208-C storage eliciting significantly greater release than either 48C or 378C (both P < 0.001, Tukey). No significant interaction was observed between the two variables (P ¼ 0.2). Predilution of blood, for ease of technical performance and to enhance supernatant yield, as expected, leads to a fall in TNFa concentration per unit supernatant with an increased ratio of media to WB ( Fig. 2A). This decrease was however not directly proportional, normalization by dilution factor failing to create equivalence between technical approaches (P < 0.01, one-way RM-ANOVA).
Considerable interindividual variation in LPS-stimulated TNFa release was observed, the supernatant of heparinized blood from 10 volunteers stimulated with 1 ng/mL S. abortus equi (6 h, 1:5 dilution, 378C, agitated) having measured concentrations ranging from 1,024 to 4,649 pg/mL (mean 2,339 pg/ mL, Fig. 2B). Expression of assayed TNFa as a function of cell number (pg/mL per 1,000 monocytes) rather than as an absolute concentration in supernatant (pg/mL) resulted in a decrease in the co-efficient of variation (39.95% vs. 47.17% respectively, Fig. 2B). The addition of a cyclooxygenase inhibitor to the assay both decreased the co-efficient of variation (37.67% vs. 47.17%) and significantly increased the measured TNFa concentration (panel A, Supplemental Figure 1

DISCUSSION
Ex vivo LPS-stimulated WB TNFa release is a biomarker of monocyte function and reflective of global immune competence, persistent reduction being predictive of adverse outcomes, and its restoration an indicator of clinical improvement (4,9,10,13). However, to date, laboratories have employed divergent techniques to determine and report this key metric (see Table 1) (14-18), impairing interpretation, comparability, and reproducibility. No clear rationale underlies these alternate approaches.
Selection of LPS source, LPS concentration, duration and temperature of incubation, anticoagulant and decision to dilute the blood varied extensively between laboratories, and were all demonstrated to significantly alter the assay result. Sample handling prior to stimulation, rarely reported in manuscripts, was additionally observed to contribute variance-both time to LPS addition and WB storage temperature-impacting subsequent TNFa release. Reporting inconsistencies further obfuscate results, studies presenting either concentration of TNFa alone (4) or expressing this as a factor of cell number (total WBC count (19), monocyte count (12)), and failing to describe the number of biological and/or technical repeats undertaken per time-point/condition per individual. Both normalizing TNFa concentration to monocyte count and pretreatment of the blood with a cyclooxygenase inhibitor were found to synergistically reduce the coefficient of variation; however, the functional and diagnostic relevance of this is unclear.
These results are not unexpected. It has long been known that varying the LPS dose or duration of incubation alters the readout of the ex vivo WB assay (14,15). Nor is it surprising that sample handling, different sources of LPS, dilution of the blood, or choice of anticoagulant alters measured TNFa concentrations. What is remarkable however is the lack of derivation and adoption of a uniform method and reporting standard for this important test of immune dysfunction: a deficiency that needs to be addressed. Stratified immunoadjuvant therapy holds great promise for critically ill patients (3,20). Establishing a panel of biomarkers with associated clinically relevant definitions and predictive value, to determine who to treat, when to treat them, and what to administer will be critical to its success (21). It is vital that avoidable methodological heterogeneity does not confound inclusion criteria, determination of outcome, and thus the external validity of future clinical trials.
It should be noted that this study was not designed to define a ''gold-standard'' method of undertaking the ex vivo WB stimulation assay, instead seeking to identify key sources of methodological variance and highlight their implications. Participants were healthy volunteers, a ''core'' method was selected and only one method of TNFa measurement was , Salmonella Minnesota R-form serotype: R595). TNFa from un-stimulated cells was undetectable. B, n ¼ 9. Different concentrations of LPS (SAE, 10 pg/mL to 100 ng/mL). C, n ¼ 10. Alternate anticoagulants: lithium heparin, sodium citrate or EDTA (all SAE, 1 ng/mL). D, n ¼ 5. For different lengths of time prior to supernatant removal (SAE, 1 ng/mL, 30 min to 24 h). E, n ¼ 5. In alternate incubation conditions either 208C or 378C, with (þ) or without (À) agitation. F, n ¼ 10. Variations in sample handling (time to stimulation [2, 4, 6, 12 h] and storage temperature [48C, 208C, 378C]). n ¼ 5 per condition. Lines denote mean and 95% confidence intervals. Differences between categorical variables were tested for using either repeated measures one-way or two-way analysis of variance with Tukey multiple comparisons test, or paired t tests. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001. EDTA indicates ethylenediaminetetraacetic acid; ns, non-significant; TNFa, tumor necrosis factor a.
performed. Response variation may have differed had an alternate system and population been employed. In particular, the importance of normalizing TNFa release to monocyte count-more variable in acute illness-to accurately gauge immune competence may have been underestimated. The blood collection tubes employed were sterile and from one batch, yet not guaranteed to be pyrogen free. Additional technical factors not elaborated here may also impact measured TNFa release including the method of LPS extraction, purification and solubilization, and the testing of either fresh or frozen supernatants. Future work may address these deficiencies and should seek to formulate and validate the predictive and diagnostic value of a standardized assay-in conjunction with additional metrics of immunosuppression-as a clinical imperative.

FIG. 2.
Variations in reporting convention of data from the ex vivo LPS-stimulated WB cytokine release assay. A, Left panel-WB (1 mL) was stimulated (SAE, 1 ng/mL) either un-diluted or prediluted with RPMI at a 1:1, 1:5, or 1:10 ratio. n ¼ 10 per condition. Right panel-measured concentrations were normalized by dilution factor to examine for equivalence. Lines denote mean and 95% confidence intervals. Differences were tested for using repeated measures one-way analysis of variance with Tukey multiple comparisons test. Statistical significance is indicated as * P < 0.05, ** P < 0.01. B, TNFa expressed as either an absolute concentration in aspirated supernatant or as a factor of cell number (/1000 monocytes). n ¼ 10. TNFa indicates tumor necrosis factor a; WB, whole blood. Eight published papers (first author, year of publication) were selected to exemplify diversity in technical performance of the assay along key variables (LPS type and concentration, use of diluent and ratio to blood, incubation period after stimulation [all at 378C], use of agitation during this period, choice of anticoagulant), sample handling prior to analysis (time and storage temperature from collection to stimulation) method of tumor necrosis factor a (TNFa) measurement and reporting of results (absolute TNFa concentration in supernatant or whether normalized to cell number). The range, mean or median (AESEM or IQR respectively) of TNFa concentrations reported in the control group in each study is also displayed. ELISA indicates enzyme-linked immunosorbent assay; N/A indicates not applicable; NI, not indicated.