HLA-DR expression in neonates after cardiac surgery under cardiopulmonary bypass: a pilot study
© The Author(s). 2018
Received: 13 October 2017
Accepted: 14 December 2017
Published: 11 January 2018
Monocyte HLA-DR expression has been reported as a marker of immunosuppression and a predictor of sepsis development. However, to date, there is no report on monocyte HLA-DR monitoring exclusively in neonates (< 28 days of life) who underwent cardiac surgery under cardiopulmonary bypass (CPB), which have a high risk of nosocomial infection. In this pilot study, we studied nine neonates with a diagnosis of congenital heart disease requiring surgery under CPB. There was a significant reduction in monocyte HLA-DR expression for the first two postoperative days, as compared to preoperatively (p = 0.004). Moreover, neonates who displayed an episode of NI had a dramatically lower HLA-DR expression at day 4, as compared to neonates without NI (4257 AB/c [2220–5895] vs 14,947 AB/c [9858–16,960]; p = 0.04). Our preliminary results could indicate that HLA-DR expression may be a useful biomarker of immunosuppression-induced secondary infection after CPB in neonates.
The pro-inflammatory response that accompanies the onset of critical illness often occurs concurrently with a compensatory anti-inflammatory response. When severe and persistent, this anti-inflammatory response has been termed immunoparalysis. The diminished monocyte human leukocyte antigen-DR (HLA-DR) expression on cell surface is proposed to reflect immunoparalysis in critically ill patients [1, 2]. To date, HLA-DR expression has been assessed in adults as a predictor of septic complications after various injuries [3–6]. In pediatric cardiac surgery, HLA-DR expression has been examined in two studies [7, 8]. However, the results were principally limited by concern related to age of patient with a large heterogeneity  or non-inclusion of children younger than 3 months . To our knowledge, no study has specifically focused on HLA-DR expression on circulating monocytes among neonates who underwent cardiac surgery under cardiopulmonary bypass (CPB), which have a high risk of nosocomial infection (NI) . In this pilot study, we investigated the kinetic of monocyte HLA-DR expression in this population and described the relationship between monocyte HLA-DR expression and the subsequent development of NI.
Blood samples were collected from neonates preoperatively at line insertion, and 1, 2, 3, and 4 days after the end of CPB on immunology laboratory working days (Monday to Friday). The number of HLA-DR molecules per monocyte (AB/c) was determined immediately after sample collection (i.e., within 90 min) by flow cytometry on whole blood using a standardized method with a Quantibrite phycoerythrin fluorescence quantitation kit (Quantibrite anti HLA-DR/Anti Monocytes CD14, BD Biosciences, Le Pont de Claix, France), as previously described [10, 11]. Blood samples for cytokine assays were immediately centrifuged, and the plasma was stored at − 80 °C. Cytokine concentrations in the plasma (IL-6, IL-8, and IL-10) were measured by multiplex immunoassay according to the manufacturer’s protocol (Merck Millipore, Molsheim, France). Immunological analysis was conducted blind, and clinical data were not available to the immunology staff before the end of the study.
NI including catheter-related bloodstream infections, ventilator-associated pneumonia, and sternal wound infections were defined based on the Center of Disease Control (CDC) and National Nosocomial Infections Surveillance criteria  and were prospectively recorded during the PICU stay or within 30 days after surgery. All parents were informed of the project and written consent was waived. The study was approved by the local ethics committee of the University Hospital of Nantes.
Statistical analyses were performed using GraphPad Prism software (GraphPad, La Jolla, CA). The Kruskal–Wallis test was used for comparisons of multiple groups (preoperatively, 1, 2, 3, and 4 days after CPB). Dunn’s multiple comparisons test was used as a post hoc test for intergroup comparisons. Continuous nonparametric variables were expressed as medians (extremes values). The Mann–Whitney test was used to compare two independent groups on day 4 (infected versus non-infected patients). Significance was defined as p-value less than 0.05.
Characteristics of patients (n = 9)
Male, n (%)
Gestational age < 36 weeks, n (%)
Genetic abnormality, n (%)
Cyanotic congenital heart defects, n (%)
Lymphocyte cell count preoperatively (/mm3)
Characteristics of surgery
RACHS-1 score a
Time on cardiopulmonary bypass (min)
Aortic cross-clamp time (min)
Hypothermia (20–28 °C), n (%)
Characteristics during the PICU stay after surgery
Use of corticoids during the first 48 h, n (%)
Extracorporeal membrane oxygenation support, n (%)
Delayed closure of sternum, n (%)
Peak Vasoactive-Inotropic score during the first 48 h b
Positive fluid balance at day 2, n (%)
Acute kidney injury at day 2 c, n (%)
Lymphocyte cell count at day 2 (/mm3)
Time on mechanical ventilation (days)
Nosocomial infection, n (%)
PICU length of stay (days)
Death, n (%)
In this pilot study, we report that neonates had a dramatic reduction in HLA-DR expression on circulating monocytes during the first two postoperative days after CPB, and those with prolonged decreased HLA-DR in the early postoperative period (day 4) could represent a subpopulation at greatly increased risk of later NI. Moreover, immunoparalysis described here after neonatal CPB accompanies a pro-inflammatory response, illustrated by high circulating levels of IL-6 and IL-8. This increase in pro-inflammatory cytokines IL-6 and IL-8 24 h after CPB is consistent with previous studies in pediatric cardiac surgery .
The main limitation of this preliminary study concerns the fact that HLA-DR values were not censored after NI diagnosis given the small number of patients included. These promising findings warrant thus a larger confirmatory trial (NCT03309839) before HLA-DR expression can be introduced in the clinical practice as a useful biomarker of immunosuppression-induced after CPB in neonates.
AC designed the study, performed the statistical analysis, and drafted the manuscript. CB and NS performed the immunological analysis. AC, NC, PB, and NJ collected the clinical data. NC, PB, NJ, RJ, AR, and KA revised it for important intellectual content. All authors approved the final version of the manuscript.
Ethics approval and consent to participate
All parents were informed of the project which was approved by the local ethics committee of the University Hospital of Nantes.
Consent for publication
Written parental consent to publish has been obtained from all children included in the study.
The authors declare that they have no competing interests.
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