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Decreased Monocyte HLA-DR Expression in Patients After Non-Shockable out-of-Hospital Cardiac Arrest

Venet, Fabienne; Cour, Martin; Demaret, Julie; Monneret, Guillaume; Argaud, Laurent

doi: 10.1097/SHK.0000000000000561
Clinical Science Aspects

ABSTRACT Out-of-hospital cardiac arrest (OHCA) constitutes a major health care problem with the development in immediate survivors of a post-cardiac arrest syndrome including systemic inflammatory response as observed in sepsis. As a decreased monocyte HLA-DR expression (mHLA-DR) has been repeatedly described in septic patients in association with an increased risk of death and nosocomial infections, we tested whether this immune alteration could also be observed after OHCA. Fifty-five non-shockable OHCA patients sampled at Day 0 (D0: within 4 h after OHCA), D1 (the next day), and D3: (after 2 additional days) were included. CD4+ lymphocyte count and mHLA-DR were evaluated by flow cytometry. We observed a marked decrease in mHLA-DR as early as D0 in patients compared with normal values. This decrease persisted till D3 and was associated with a moderate decrease in the number of circulating CD4+ lymphocytes. No correlations were identified between mHLA-DR and usual prognostic markers after OHCA. However, overtime evolution in mHLA-DR values appeared different between survivors and non-survivors with a quasisystematic decrease between D1 and D3 in non-survivors versus an increased expression in survivors. In conclusion, this preliminary pilot study describes the occurrence of OHCA-induced immune alterations as illustrated by a decreased mHLA-DR and CD4+ lymphopenia. Furthermore, we show for the first time the differential overtime evolution in mHLA-DR between survivors and non-survivors without association with usual prognostic markers and multiple organ failure. These initial results should now be confirmed in a larger cohort of OHCA patients.

*Groupement Hospitalier Edouard Herriot, Cellular Immunology Laboratory

Université Claude Bernard Lyon I and Hospices Civils de Lyon, EAM 4174, Lyon, France

Medical Intensive Care Unit, Groupement Hospitalier Edouard Herriot, Hospices Civils de Lyon

§INSERM UMR 1060, CarMeN, Team 5 “Cardioprotection”, Lyon, France

Address reprint requests to Prof Laurent Argaud, MD, PhD, Service de Réanimation Médicale, Groupement Hospitalier Edouard Herriot, 5, place d’Arsonval, 69437 Lyon Cedex 03, France. E-mail:

Received 3 November, 2015

Revised 23 November, 2015

Accepted 4 January, 2016

FV, MC, LA, GM conceived of the study, and participated in its design and coordination. MC and LA included patients. JD carried out the immunoassays. FV wrote the manuscript and performed the statistical analysis. MC, LA, GM helped to draft the manuscript. All authors read and approved the final manuscript.

The authors report no conflicts of interest.

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Out-of-hospital cardiac arrest (OHCA) constitutes a major public health problem with hundreds of thousands deaths occurring every year in Western countries. Despite improvements in cardiopulmonary resuscitation, mortality remains unacceptably high (up to 95%) (1). Among OHCAs, those with a non-shockable cardiac rhythm are both the most frequent and have the poorest prognosis (mortality up to 95%) (1). Most immediate survivors subsequently die of the consequences of the global ischemia-reperfusion insult, including brain injury and cardiovascular failure (2).

This post-cardiac arrest syndrome also includes the development of a systemic inflammatory response syndrome recalling observations made in severe septic patients (2). Interestingly, as in septic shock, non-shockable OHCA is particularly at risk of developing multiple organ failure (3). In sepsis, it is now accepted that this initial pro-inflammatory response is associated with the secondary development of immune alterations which intensity and duration have been shown to be associated with an increased risk of death or nosocomial infections (4). Among these, the decreased expression on circulating monocytes of the major histocompatibility complex II molecule HLA-DR (mHLA-DR) has been repeatedly described in septic patients (5). To our knowledge, there is no data available regarding standardized mHLA-DR expression after OHCA.

In this study, we therefore explored mHLA-DR expression on circulating monocytes in a cohort of patients after non-shockable OHCA.

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Study design

This study was approved by the local medical ethics committee (Comité de Protection des Personnes Sud-Est II), which waived the need for informed consent because this study was observational and biomarkers expressions were measured on residual blood after completion of routine follow-up. This study is also registered at French Ministry of Research and Teaching and recorded at the Commission Nationale de l’Informatique et des Libertés.

The study population consists of adult patients admitted, from January 2011 to July 2014, to our medical intensive care unit (ICU) after non-shockable OHCA. All patients were treated according to the 2010 International Consensus on Cardiopulmonary Resuscitation (6). All patients received therapeutic hypothermia with the use of surface cooling techniques.

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Data collection

Cardiac arrest was characterized according to the Utstein style (7). Clinical and biological data were recorded after patients’ inclusion in the protocol. Data collection included demographic characteristics, comorbidities, cause, and characteristics of cardiac arrest. Based on the worst data from the first 24 h after hospital admission, two clinical scores were recorded: the initial severity assessed by the Simplified Acute Physiology Score II (SAPS II) and the Sepsis-related Organ Failure Assessment (SOFA) score (8, 9). Mortality was defined as death occurring within 28 days after hospital admission.

Biological analyses were performed on residual blood after completing routine follow-up performed in the ICU. EDTA-anticoagulated blood was collected from patients at three time points on Immunology Laboratory working days (Monday to Friday): day 0 (D0, i.e., within the first 4 h after OHCA), day 1 (D1, at 24 ± 4 h after OHCA), and day 3 (D3) after OHCA.

Monocyte HLA-DR expression was assessed by flow cytometry (Navios; Beckman-Coulter, Miami, FL) using standardized technique, as previously described (10). Briefly, whole blood (50 μL) was stained with 20 μL of QuantiBrite HLA-DR/monocyte mixture (QuantiBrite anti-HLA-DR PE (clone L243)/anti-monocytes (CD14) PerCP-Cy5.5 (clone MϕP9), Becton Dickinson, San Jose, CA). Results are expressed as number of antibodies bound per cells (AB/C) thanks to calibrated PE-beads (BD Quantibrite- PE Beads, Becton Dickinson). Time-to-results for mHLA-DR measurement was less than 1 h and cost was about 10 € (around 10 USD). Absolute lymphocyte and neutrophil counts were measured at D0, D1, and D3; number of circulating CD4+ T cells at admission was also reported. Monoclonal antibodies were used according to the manufacturer's recommendations: FITC-labeled anti-CD45, PC5-labeled anti-CD3, PE-labeled anti-CD4 (Beckman-Coulter). The red cells were lysed on the automated TQ Prep system (Beckman-Coulter). Absolute count was performed using Flow-count fluorospheres according to manufacturer's recommendations (Beckman-Coulter) (11, 12).

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Statistical analysis

Results are presented as box-plots and individual values. Data are expressed as medians and interquartile ranges [Q1–Q3]. Non-parametric Wilcoxon paired-test was performed to evaluate overtime evolution in patients with multiple sampling. Non-parametric Mann–Whitney U test was used to compare results between survivors and non-survivors. Spearman tests were used to assess correlations. Statistical analyses were performed using GraphPad Prism Software (version 5.03, GraphPad, La Jolla, CA). A P value lower than 0.05 was considered significant.

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Clinical and demographic characteristics of the 55 patients with OHCA are presented in Table 1. The high severity of this cohort is illustrated by the elevated values of SAPS II and SOFA scores and the high mortality (91%; n = 5 survivors).

Table 1

Table 1

Immediately after OHCA (D0), patients presented with a markedly decreased mHLA-DR (Fig. 1), when compared with standardized normal values (>20,000 AB/C) (13). This was associated at ICU admission with a moderate decrease in the number of circulating CD4+ cells (366 [215–553] cells/μL), while total lymphocyte count was normal (3.0 [1.3–4.9] G/L) and with a marked increase in the number of circulating neutrophils (12.1 [7.2–17.9] G/L). Regarding overtime evolution, we observed that mHLA-DR continued to decrease at D1 and remained low at D3 after OHCA but with no further decrease. Indeed, we observed a significant decrease in mHLA-DR between D0 and D1 (P < 0.001) but no difference between D1 and D3 (P = 0.29). We did not observe any difference in mHLA-DR (P = ns) between male and female at any time point.

Fig. 1

Fig. 1

When comparing mHLA-DR values in survivors and non-survivors versus normal values, we observed a similarly decreased mHLA-DR (P = ns) at every time point in both groups (Fig. 2). While an early lymphopenia was observed (D1: 0.8 [0.5–1.2] G/L, D3: 1.0 [0.7–1.2] G/L, P < 0.01 vs. D0), total lymphocyte count did not differ between survivors and non-survivors at D1 (1.1 [0.7–1.2] vs. 0.8 [0.5–1.2] G/L, P = 0.58), and D3 (1.1 [1.0–1.2] vs. 1.0 [0.7–1.2] G/L, P = 0.52). Neutrophil number was also similar between survivors and non-survivors (P = ns). Most interestingly, overtime evolution of mHLA-DR appeared different between survivors and non-survivors. Indeed, we observed a significant decrease in mHLA-DR between D0 and D1 in non-survivors (n = 16, P < 0.001), but not in survivors (P = 0.63, Fig. 2). In addition, mHLA-DR continued to decrease between D1 and D3 in non-survivors (P < 0.05) while it tends to increase in survivors (P = ns). This is best illustrated when comparing overtime evolution of mHLA-DR in 10 patients with 3 consecutive samples (Fig. 3), which illustrates the quasisystematic decrease in mHLA-DR in non-survivors (n = 6/7, 86%) versus the increased expression in survivors (n = 3/3, 100%) between D1 and D3 after OHCA (P < 0.05).

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Finally, in studying the whole cohort, we did not find any correlation at any time point between mHLA-DR and usual markers of OHCA severity such as no-flow and/or low-flow duration, dose of epinephrine, SAPS II, or SOFA scores (data not shown).

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This preliminary pilot study describes the decreased mHLA-DR and CD4+ lymphopenia after non-shockable OHCA. Furthermore, we show for the first time, based on standardized measurement, the differential overtime evolution in mHLA-DR between survivors and non-survivors without association with usual prognostic markers and multiple organ failure.

Post-cardiac arrest syndrome is a complex and only partially elucidated process that culminates in multiple organ failure (2, 3). It is characterized by the occurrence of an ischemia-reperfusion syndrome and the non-specific systemic activation of the immune response (2). This condition has many features in common with sepsis (14). Indeed, as early as 3 h after cardiac arrest, blood concentrations of various cytokines, soluble receptors, and endotoxin increase and the magnitude of these changes is associated with outcome (14). In the current study, we further extend these results by showing in a cohort of 55 patients the decreased mHLA-DR and reduced circulating CD4+ T cell number after non-shockable OHCA. Consensus exists that decreased mHLA-DR expression serves as a surrogate marker of monocyte anergy (4, 5). Several studies have demonstrated an association of low mHLA-DR with altered monocyte functions, e.g., lower tumor necrosis factor α and interleulin-1β release in response to lipopolysaccharide challenges, and decreased lymphocyte proliferation in response to recall antigens (due to impaired antigen presentation). Most importantly, decreased mHLA-DR expression is associated with increased risk of nosocomial infections and death (4, 5). The present study reproduces observations made after septic shock and suggests that, in addition to systemic inflammation, compensatory immune mechanisms are induced as well during post-cardiac arrest syndrome (4). Indeed, we observed here that, at D1, almost 80% of OHCA patients presented with mHLA-DR values lower than 10,000 AB/C, which corresponds to a severe immunodepression based on previous work using this standardized technique of mHLA-DR measurement (13). This is also very similar to values observed after septic shock or in severe trauma patients (10, 15). Finally, this is in accordance with previous works showing, in small cohorts of patients, the blunted host immune response to toll-like receptor ligands and the dysregulated production of cytokines after cardiac arrest (16–18). While a decrease in mHLA-DR after OHCA was previously reported, it is now confirmed in a larger cohort of non-shockable cardiac arrest patients with a standardized measurement of mHLA-DR (16).

Interestingly, the extent of this immunosuppression evaluated by mHLA-DR appears independent of usual mortality prognostic markers and of multiple organ failure after OHCA. In addition, overtime evolution appeared different between survivors and non-survivors, with non-survivors presenting with persistent mHLA-DR decrease overtime while mHLA-DR values in survivors increased between D1 and D3 after OHCA. Similar differences in mHLA-DR evolution between survivors and non-survivors were, for example, made after ruptured abdominal aortic aneurysms (19). However, mHLA-DR could probably be used as a surrogate marker of the severity of the post-cardiac arrest syndrome but not as a relevant prognostic factor in this field. Thus, mHLA-DR evaluation may represent a novel standardized monitoring tool after OHCA, affordable and readily available in the clinical routine. In particular, it could help clinicians to evaluate the consequences of the systemic ischemia-reperfusion injury and the impact of therapeutic management on immunosuppression after OHCA, independently of multiple organ failure. This is all the more relevant since it has been suggested in other clinical contexts that immunosuppression may participate in an increased risk of nosocomial infections (5, 20). As early infectious complications are frequent in post-OHCA patients since up to two-third of these patients will present with a septic episode within the first 3 days after ICU admission, mHLA-DR measurements at these time points may also represent a new tool to assess the post-OHCA infectious risk (21, 22). For instance, low mHLA-DR could prompt physicians to perform additive bacteriological sampling and/or to initiate early antibiotic treatment. Indeed, mHLA-DR directly measures monocyte anergy and altered functionality and is considered the best marker to date of injury-induced immunosuppression (4, 5). Thus, our study opens up the possibility of using mHLA-DR as a reasonable alternative endpoint to test interventions targeting the post-cardiac arrest syndrome. Obviously, our study has at least one limitation. As a preliminary pilot work, the low number of patients (and their severity) precludes us to perform robust statistical analyses (especially on infectious complications) but these first promising results deserve now to be extended to a larger cohort of patients in a multicentered study.

In summary, our results suggest that OHCA patients develop cardiac arrest-induced immune alterations as illustrated by a decreased mHLA-DR and CD4+ lymphopenia. We also present here preliminary results showing the differential overtime evolution in mHLA-DR between survivors and non-survivors without association with initial prognosis factors after OHCA.

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The authors thank Anne Portier (Immunology Laboratory) as well as Sylvie De La Salle (Medical ICU) for their respective great help in experiments performing, clinical data gathering. The authors are also very grateful to Dr Delphine Maucourt-Boulch (Department of Medical Statistics, Hospices Civils de Lyon) for statistical advices.

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Immunodepression; lymphopenia; monocyte HLA-DR; out-of-hospital cardiac arrest; post-cardiac arrest syndrome

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