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Prospective flow cytometry analysis of leucocyte subsets in critically ill patients who develop sepsis: a pilot study

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Abstract

Purpose

Sepsis in critically ill patients with injury bears a high morbidity and mortality. Extensive phenotypic monitoring of leucocyte subsets in critically ill patients at ICU admission and during sepsis development is still scarce. The main objective of this study was to identify early changes in leukocyte phenotype which would correlate with later development of sepsis.

Methods

Patients who were admitted in a tertiary ICU for organ support after severe injury (elective cardiac surgery, trauma, necessity of prolonged ventilation or stroke) were sampled on admission (T1) and 48–72 h later (T2) for phenotyping of leukocyte subsets by flow cytometry and cytokines measurements. Those who developed secondary sepsis or septic shock were sampled again on the day of sepsis diagnosis (Tx).

Results

Ninety-nine patients were included in the final analysis. Nineteen (19.2%) patients developed secondary sepsis or septic shock. They presented significantly higher absolute monocyte counts and CRP at T1 compared to non-septic patients (1030/µl versus 550/µl, p = 0.013 and 5.1 mg/ml versus 2.5 mg/ml, p = 0.046, respectively). They also presented elevated levels of monocytes with low expression of L-selectin (CD62Lneg monocytes) (OR[95%CI] 4.5 (1.4–14.5), p = 0.01) and higher SOFA score (p < 0.0001) at T1 and low mHLA-DR at T2 (OR[95%CI] 0.003 (0.00–0.17), p = 0.049). Stepwise logistic regression analysis showed that both monocyte markers and high SOFA score (> 8) were independently associated with nosocomial sepsis occurrence. No other leucocyte count or surface marker nor any cytokine measurement correlated with sepsis occurrence.

Conclusion

Monocyte counts and change of phenotype are associated with secondary sepsis occurrence in critically ill patients with injury.

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Availability of data and materials

The datasets used and/or analysed (beyond those included in the supplementary files) during the current study are available from the corresponding author on reasonable request.

Abbreviations

AUC:

Area under the curve

BSI:

Blood stream infection

CI:

Confidence interval

CLABSI:

Central line associated blood stream infection

CRP:

C-reactive protein

HAP:

Hospital-acquired pneumonia

HLA-DR:

Human leucocyte antigen

ICU:

Intensive care unit

IQR:

Interquartile range

LOS:

Length of stay

MFI:

Median fluorescence index or median of fluorescence intensity

OR:

Odds ratio

PE:

Phycoerythrin-linked

PerCP:

Perinidin-chlorophyll protein-linked

ROC:

Receiver operating characteristic

SOFA:

Sequential organ failure assessment

SSTI:

Surgical site and soft tissue infection

TNF-α:

Tumor necrosis factor α

VAP:

Ventilator-associated pneumonia

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Acknowledgements

We are grateful for the insightful revision of Profs B. Misset and JM Cavaillon. We are thankful to BD Sciences for the generous gift of monoclonal antibodies.

Funding

Not applicable.

Author information

Authors and Affiliations

Authors

Contributions

NL, PD, CO and AG designed the study; NL, CG, CD, CL, AH and AG did the experiments; NL, CG, NM and AG analysed the data; NL wrote the manuscript, CG and AG revised the manuscript.

Corresponding author

Correspondence to Nathalie Layios.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Ethics approval and consent to participate

The study was appointed the Belgian number B707201111981 by the local ethics committee of University Hospital of Liège (number 707) and written informed consent was obtained from the patient or his/her legal representative.

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Not applicable.

Supplementary Information

Below is the link to the electronic supplementary material.

15010_2023_1983_MOESM1_ESM.jpg

Supplementary file1 Figure S1 (A-J) Flow cytometry gating strategy used to define lymphocyte, monocyte and granulocyte subsets by flow cytometry (explanation in the text). (JPG 246 KB)

15010_2023_1983_MOESM2_ESM.jpg

Supplementary file2 Figure S2 Panel A: Measurements at ICU admission in nonseptic and septic patients and in healthy controls (> 50 years). (*: p<0.05) (JPG 90 KB)

15010_2023_1983_MOESM3_ESM.jpg

Supplementary file3 Figure S2 Panel B: Predictive value of monocyte absolute count (/µl) obtained at T1. ROC curve analysis of sepsis occurrence based on levels of monocytes and of SOFA is shown. (JPG 109 KB)

15010_2023_1983_MOESM4_ESM.jpg

Supplementary file4 Figure S3 Absolute count of CD62Lneg monocytes (/µl): evolution of septic patients (N=12 patients with measurement at ICU admission, 48 to 72h later and on the day of sepsis diagnosis). (ns: not statistically significant). (JPG 86 KB)

15010_2023_1983_MOESM5_ESM.jpg

Supplementary file5 Figure S4 Intermediate monocytes (CD14++CD16+) median HLA-DR (MFI): evolution of septic patients (N=7 with measurement at ICU admission, 48 to 72h later and on the day of sepsis diagnosis). (ns: not statistically significant). (JPG 87 KB)

15010_2023_1983_MOESM6_ESM.docx

Supplementary file6 Table S1 Sites of infection and microbiological documentation. HAP-VAP: hospital-acquired pneumonia. VAP: ventilator-associated pneumonia. SSTI: surgical site and soft tissue infection. CLABSI: central line associated blood stream infection. BSI: primary blood stream infection. Some patients developed more than one infection and some infections were polymicrobial. Two episodes of VAP were clinically diagnosed and empirically treated although no organism grew in culture. (DOCX 17 KB)

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Layios, N., Gosset, C., Maes, N. et al. Prospective flow cytometry analysis of leucocyte subsets in critically ill patients who develop sepsis: a pilot study. Infection 51, 1305–1317 (2023). https://doi.org/10.1007/s15010-023-01983-3

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