EARLY PERSISTENT LYMPHOPENIA AND RISK OF DEATH IN CRITICALLY ILL PATIENTS WITH AND WITHOUT SEPSIS

ABSTRACT Purpose: To examine the relationship of early persistent lymphopenia with hospital survival in critically ill patients with and without sepsis to assess whether it can be considered a treatable trait. Methods: Retrospective database analysis of patients with nonelective admission to intensive care units (ICUs) during January 2015 to December 2018. Patients were classified as having sepsis if the Acute Physiology and Chronic Health Evaluation III admission diagnostic code included sepsis or coded for an infection combined with a Sequential Organ Failure Assessment score of ≥2. We defined early persistent lymphopenia at two thresholds (absolute lymphocyte count [ALC] <1.0 and <0.75 × 109/L) based on two qualifying values recorded during the first 4 days in ICU. The main outcome measure was time to in-hospital death. Results: Of 8,507 eligible patients, 7,605 (89.4%) had two ALCs recorded during their first 4 days in ICU; of these, 1,482 (19.5%) had sepsis. Persistent lymphopenia (ALC <1.0) was present in 728 of 1,482 (49.1%) and 2,302 of 6,123 (37.6%) patients with and without sepsis, respectively. For ALC <0.75, the results were 487 of 1,482 (32.9%) and 1,125 of 6,123 (18.4%), respectively. Of 3,030 patients with persistent lymphopenia (ALC <1.0), 562 (18.5%) died compared with 439 of 4,575 (9.6%) without persistent lymphopenia. Persistent lymphopenia was an independent risk factor for in-hospital death in all patients. The hazard ratios for death at ALC <1.0 were 1.89 (95% confidence interval, 1.32–2.71; P = 0.0005) and 1.17 (95% confidence interval, 1.02–1.35; P = 0.0246) in patients with and without sepsis respectively. Conclusions: Early persistent lymphopenia is common in critically ill patients and associated with increased risk of death in patients with and without sepsis. Although the association is stronger in patients with sepsis, lymphopenia is a candidate to be considered a treatable trait; drugs that reverse lymphopenia should be trialed in critically ill patients.


INTRODUCTION
Persistent lymphopenia is known to be associated with increased mortality in critically ill patients with sepsis (1) and trauma (2)(3)(4), but whether this association holds for other critically ill patients is unknown.Patients who survive critical illness often experience long-term morbidity affecting physical function, cognition, and mental health (5,6), and these long-term health effects are similar in patients with and without sepsis (7).The terms postintensive care syndrome and postsepsis syndrome describe these persisting adverse health effects.In addition, readmission rates to hospital for sepsis survivors are high.In high-income countries, around 40% of sepsis survivors are readmitted to hospital within 90 days, with recurrent sepsis being a common reason for readmission.This supports the hypothesis that sepsis survivors experience persistent immunosuppression (8).In patients with sepsis, multiple factors influence the immune system, including the microbiome (9), which affects the postsepsis metabolic and epigenetic immune composition (9,10).This results in complex alterations to cellular immunity with consequent immunodeficiency.The immune dysfunction, which can be predicted by serum-soluble programmed cell death ligand-1 concentration (11), can often be prolonged (12)(13)(14)(15) and is postulated to be a leading cause of late mortality in patients who have survived sepsis (13,16).
Published data on the prevalence and prognostic value of persistent lymphopenia in critically ill patients without sepsis or trauma are scarce.The longer-term outcomes of critically ill patients in general and those with sepsis are similar.Therefore, reversal of lymphopenia is a potential strategy to reduce the risk of death and longer-term morbidity in critically ill patients regardless of their sepsis status (17,18).We hypothesized that early persistent lymphopenia would be common and associated with an increased risk of death in critically ill patients without sepsis making it potentially a treatable trait, an observable biologic abnormality potentially present in different disease states whose modification results in improved outcomes (19).
Consequently, we analyzed data from two intensive care units (ICUs) in Queensland, Australia, to examine the relationship of persistent lymphopenia with risk of death and other outcomes in critically ill patients with and without sepsis.The present study represents one of the largest studies to date examining the effect of lymphopenia on mortality in critically ill patients with and without sepsis.

Study setting and population
Ethics approval for data collection and analysis was granted by Metro South, Human Research Ethics Committee, Queensland, Australia.We interrogated the clinical information datasets (iMDsoft, MetaVision, Park Atidim, Tel Aviv, Israel) from the ICU of two tertiary hospitals (total of 2,019 beds) in Queensland, Australia.
We accessed data for adult patients admitted to the study ICUs between January 2015 and December 2018.We defined early persistent lymphopenia using two threshold values: an absolute lymphocyte count (ALC) <1.0 Â 10 9 and <0.75 Â 10 9 /L, recorded on at least 2 days during the first 4 days in the ICU.Patients admitted to ICU after elective surgery were excluded as they were expected to have a short ICU stay and a low risk of death.
We used the Acute Physiology and Chronic Health Evaluation (APACHE) III (20) admission diagnostic code to classify the patient as having sepsis or not.We classified patients as having sepsis if the admission diagnostic code specified sepsis or the admission diagnosis was infection or pathology consistent with a diagnosis of sepsis (e.g.perforated bowel), combined with a Sequential Organ Failure Assessment (SOFA) score ( 21) of 2 or more.Patients younger than 18 years and/or with a diag-nosis of lymphoma, leukemia, or immunosuppression, including HIV/AIDS, were also excluded.

Data collection and storage
Individual datasets for eligible participants were extracted, deidentified, and securely stored.These included general demographics, primary diagnosis, and comorbidities, observational variables measured within the first 24 h in the ICU.

Outcomes
The primary outcome is in-hospital death in patients with a recorded ALC <1.0 on at least 2 days within the first 4 days of ICU admission.Secondary outcomes were the proportion of patients with or without sepsis having early persistent lymphopenia defined at the two thresholds, the association of early persistent lymphopenia at the two thresholds with hospital mortality, and its association with use and duration of organ support (ventilation, vasopressor treatment, and renal replacement therapy [RRT]), and with length of stay in the hospital and ICU.

Statistical analysis
Data were analyzed by the Statistical Services Division of The George Institute for Global Health, Australia, according to a prespecified statistical analysis plan.For continuous variables, we report mean and standard deviation (SD) or medians and interquartile ranges (IQR) as appropriate.For categorical variables, we report proportions, with 95% confidence intervals (CIs) calculated by Wilson method.
We assessed time to in-hospital death using uni-and multivariable Cox regression models.The multivariable models included a set of covariates that were defined a priori with no automatic selection; these were age, sex, sepsis, early persistent lymphopenia, presence of comorbidities, SOFA score, and quartiles of illness severity using the APACHE III score.
We checked the proportional hazard assumption by visual assessment of the log cumulative-hazard functions and by Kolmogorov-type Supremum test.We fitted hierarchical logistic models for binary outcomes (such as treatment with ventilation, RRT, or vasopressors), and for length of ICU and hospital stay, we used mixed models with the same covariates described previously.For subgroup analyses, we explored by septic/nonseptic patients and lymphopenia presence/absence using the two thresholds prespecified (<1.0 and <0.75 Â 10 9 /L).We performed complete case analyses with statistical significance set at alpha of 0.05, and we made no adjustments for multiple comparisons.We used SAS v9.3 (Cary, North Carolina, USA) for all statistical analyses.

Patients
We identified 8,507 potentially eligible patients, of whom 902 (10.6%) were excluded as they did not have two ALCs recorded within the first 4 days of admission; 848 of the 902 (94.01%) had an ICU stay of less than 48 h, leaving 7,605 eligible patients (Fig. 1).

Association of persistent lymphopenia with length of stay in hospital
In all patients, early persistent lymphopenia (ALC <1.0 Â 10 9 /L) was associated with an increase in hospital length of stay of 5.41 days (95% CI, 4.05-6.77;P < 0.0001) compared with patients without early persistent lymphopenia.Patients without sepsis had an increase in hospital stay of 6.18 days (95% CI, 4.63-7.73;P < 0.0001).For patients with sepsis, the increase was 1.79 days (95% CI, −1.04 to 4.62; P = 0.2152) (Table 3a).The results using the ALC cut-off of 0.75 Â 10 9 /L were similar; patients without sepsis had an increased in hospital stay of 5.80 days (95% CI, 3.88-7.23;P < 0.0001) compared with patients with sepsis who had an increased in hospital stay of 1.52 days (95% CI, −1.47 to 4.50; P = 0.3184) (Table 3b).

DISCUSSION
The current study represents one of the largest studies to date that examines the association of lymphopenia with survival in critically ill patients with and without sepsis.We found that early persistent lymphopenia, defined as an ALC <1.0 Â 10 9 /L on at least two of the first 4 days in ICU, was common in critically ill patients both with and without sepsis.The likelihood of having early persistent lymphopenia was significantly greater in patients with sepsis; this is consistent with the trend observed by Andreu-Ballester et al. (22) in a retrospective study conducted at two hospitals in Spain.This difference was greater for patients with an ALC <0.75 Â 10 9 /L.Early persistent lymphopenia was associated with an increased risk of in-hospital death; again, this association was stronger for patients with sepsis.
For secondary outcomes, early persistent lymphopenia was associated with increased likelihood of treatment with mechanical ventilation, vasoactive drugs, and RRT.In the population studied, these findings resulted from a strong association for patients without sepsis.Early persistent lymphopenia was associated with increased length of ICU and hospital length of stay.The increase in hospital length of stay in those with lymphopenia was much greater for patients without sepsis: this was due to a shorter length of stay for patients who have neither sepsis nor lymphopenia.
We observed that early persistent lymphopenia is an independent risk factor for in-hospital death in patients with and without sepsis.Although this association has been reported for patient with sepsis (1,23), we are not aware of it being reported in a heterogeneous population of critically ill patients without sepsis.
The strengths of our study include its being multicenter and including data from over 7,000 patients.This adds very substantially to previously published data (1).We excluded patients admitted to the ICU after elective surgery, focusing our study on a cohort at greater risk of death.In addition, we selected a patient-centered primary outcome, preselected a broad set of clinically relevant variables for multivariate analysis, and followed a prespecified statistical analysis plan.Our data were obtained from two tertiary centers in Queensland, Australia, where daily blood sampling routinely includes a differential white cell count.Consequently, only 54 of the 902 (6%) patients excluded from the study were excluded because of missing lymphocyte counts.We therefore have confidence that the Australian data provide a reliable estimate of the proportion of patients with and without sepsis who exhibit early persistent lymphopenia in the Australian healthcare setting.
Limitations of our study include that we classified patients as having sepsis or not based on admission APACHE III coding and were not able to identify patients who developed sepsis later in the ICU stay.This may have reduced apparent differences between patients classified as having or not having sepsis.In addition, our data come from two similar Australian hospitals, and they may not be representative of data in other settings.
Our study was conceived as part of a planning process for a clinical trial to determine whether reversing lymphopenia will improve outcomes in critically ill patients.Given lymphopenia is associated with an increased risk of death in critically ill patients with and without sepsis, it can be used for prognostic enrichment of clinical trials by selecting a population of patients at increased risk of dying.Whether it is a treatable trait and can therefore be used for predictive enrichment to select patients more likely to respond to particular treatments is currently unknown.Treating lymphopenia in a diverse population of critically ill patients aligns with the current trend to look for treatable traits (19).However, given the stronger association of lymphopenia with risk of death in patients with sepsis, these patients may be considered the most appropriate population in which to conduct first trials of treatments designed to reverse lymphopenia.Copyright © 2024 by the Shock Society.Unauthorized reproduction of this article is prohibited.

CONCLUSION
Persistent lymphopenia is common in critically ill patients with and without sepsis and is associated with increased risk of death with the strength of the association being greater in patients with sepsis.Trials designed to examine the effect of reversing lymphopenia are warranted.

TABLE 1 .
Characteristics of patients according to classification as sepsis or not †APACHE II score ranges from 0 to 71, with higher scores indicating increased risk of death.‡ APACHE III score ranges from 0 to 299, with higher scores indicating increased risk of death.¶ SOFA integer score ranges from 0 to 24, with higher scores indicating a greater degree of organ dysfunction.f indicates frequency.Persistent lymphopenia was defined as an ALC <1.0 Â 10 9 /L and <0.75 Â 10 9 /L, on at least 2 days within the first 4 days in ICU.

TABLE 3A .
Length of ICU and hospital stay, occurrence and duration of advanced organ support comparing patients with and without sepsis and with and without persistent lymphopenia defined by absolute lymphocyte threshold of <1.0 Â 10 9 /L Secondary outcomes for patients with sepsis and without sepsis (ALC <1.0 Â 10 9 /L) *difference, †OR.

TABLE 3B .
Length of ICU and hospital stay, occurrence and duration of advanced organ support comparing patients with and without sepsis and with and without persistent lymphopenia defined by absolute lymphocyte threshold of <0.75 Â 10 9 /L Secondary outcomes for patients with and without sepsis (ALC <0.75 Â 10 9 /L) ALC, absolute lymphocyte count; CI, confidence interval; OR, adjusted odds ratio.202 SHOCK VOL.61, NO. 2 ADIGBLI ET AL.