Nucleated Red Blood Cells for Predicting Mortality in Intensive Care Unit Patients With Sepsis

Introduction

Sepsis is an irregular and complex response of the body to infection. Early initiation of antibiotherapy is of vital importance, and organs and hemodynamics should be stabilized and supported with appropriate hydration and vasoactive drugs [1,2]. The complex pathophysiology of sepsis remains a key area of research. Even with the development of modern medicine, sepsis remains one of the leading causes of death worldwide, although many advances have been made in diagnosis and treatment [3]. Although the importance of early diagnosis and treatment is recognized, many patients experience diagnostic uncertainty and delays, and thus do not receive appropriate treatment [4].

Although systemic symptoms and organ dysfunctions guide clinicians, they are not as sensitive and specific as pathogen-specific markers [5]. One of the most important points in sepsis is to differentiate the clinical picture from that of non-infectious inflammation by revealing possible pathogens and to start the most appropriate treatment [4]. Culture screening takes several days, which leads to a loss of valuable time for the patient. Pathogen-specific markers, such as direct antigen tests, allow early treatment by detecting the pathogen. However, false-negative results according to serogroups and low sensitivity require clinicians to be cautious in some cases [3]. Therefore, the search for a biomarker or scoring system that predicts sepsis steadily continues [6,7].

Many studies continue to be conducted on the clinical outcomes, course, morbidity, and mortality of sepsis, a condition that can lead to a severe clinical picture, multiple organ failure, and even death [3,6,8]. Until now, the relationship of the disease with almost all parameters, especially neutrophil-to-lymphocyte ratio, immature granulocytes, interleukins, procalcitonin, C-reactive protein (CRP), and scoring systems such as the Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) score has been investigated [1,6,9–13]. Although lactate has an important place in the detection of septic shock in the Sepsis-3.0 criteria, it is included both alone and in combination with different parameters in studies. Lactate level is accepted as an indicator for mortality [4,13]. SOFA and quick SOFA (qSOFA) scores are used to indicate the severity of organ dysfunction. The scores have secured their place in sepsis guidelines because they are more objective and have higher specificity than previously used systemic inflammatory response criteria. They are valuable in predicting patients at high risk and provide a warning for early management of organ dysfunction in sepsis [2,4].

Nucleated red blood cells (NRBCs) are erythrocyte precursors found in the blood of the fetus and early neonate [14]. They lose their nuclei and follow the reticulocyte and mature erythrocyte cycle, respectively. NRBCs are not typically seen in healthy individuals. It has been reported that their numbers increase and bone marrow functions are affected in conditions such as blood loss, hypoxia, infection, sepsis, and stress [15]. For these reasons, NRBCs have been reported as a prognostic marker in the early prediction of sepsis in certain patients [16].

In light of this information, in this retrospective study, we aimed to evaluate the NRBC count and percentage to examine their effects on the clinical outcomes and mortality of patients with sepsis followed in the intensive care unit (ICU).

Material and Methods

ETHICAL CONSIDERATIONS:

This retrospective study was performed after receiving approval from the Ankara Etlik City Hospital Scientific Research Evaluation and Ethics Committee on May 8, 2024 (No. AEŞH-BADEK 2024–248).

STUDY DESIGN AND POPULATION:

The data of the study were obtained from patients with sepsis followed in the ICU of Ankara Etlik City Hospital between March 1, 2023, and March 1, 2024. All investigators accepted and approved the principles of compliance with the Declaration of Helsinki.

Patients were excluded if they stayed in the ICU for less than 48 hours; were under 18 years of age; had trauma; were in hemorrhagic shock and received blood product transfusions before ICU admission; were receiving erythropoietin therapy; were pregnant; had hematologic diseases such as chronic myeloid leukemia, myelodysplastic syndrome, or thalassemia syndromes; had malignancies; or had incomplete medical data.

The source of infection was documented, and microbiological culture results were recorded to identify causative organisms. In addition to the presence of infection, patients with a respiratory rate of 22 or more breaths per minute, a Glasgow Coma Scale (GCS) score of less than 15, and a qSOFA score of 2 or more, with a systolic blood pressure of less than 100 mmHg were evaluated for sepsis; those with an increase of more than 2 in the SOFA score, which evaluates respiratory, coagulation, liver, cardiovascular, central nervous system, and renal system organ dysfunction, compared with baseline, were considered septic. In the SOFA scoring system, each organ system is given a score between 0 (normal function) and 4 (severe dysfunction), and the total score reflects the overall severity of organ dysfunction. In the absence of hypovolemia, those patients requiring vasopressors to maintain a mean arterial pressure of 65 mmHg or higher or those with lactate levels above 2 mmol/L were considered to have septic shock. For patients with sepsis-related hypoperfusion or septic shock, we adhered to the administration of at least 30 mL/kg intravenous (IV) crystalloid fluid within the first 3 hours of resuscitation, taking into account additional comorbidities [1,2]. Patients who did not receive blood transfusions prior to ICU admission were included in the study. Patients were categorized according to the etiology of sepsis: pneumosepsis, urosepsis, soft tissue, gastrointestinal, and catheter-related. Patients with sepsis of unknown cause or source of infection were not included in the study. Only patients who were septic at the time of ICU admission were included. Patients were followed up until they were discharged from the hospital after the ICU, and those who required multiple intensive care follow-ups were excluded from the study. Mortality in the ICU was determined as the primary outcome.

DATA COLLECTION:

Patients’ sex, age, diagnosis and etiology, comorbidities, laboratory values, ICU length of stay, total hospital length of stay, disease outcome (alive or deceased), and GCS score were obtained from patient observation forms and the hospital information management system. The need for vasopressors, the requirement for invasive mechanical ventilation, the development of acute kidney injury, and whether renal replacement therapy (intermittent or continuous) was administered were recorded. Biochemical parameters, complete blood count, and arterial blood gas analysis were performed upon admission to the ICU. These examinations were also recorded daily throughout the patient’s ICU stay. All blood count parameters were measured using a Sysmex XN blood analyzer (Sysmex, Kobe, Japan). The NRBC count and percentage were automatically measured by this analyzer using flow cytometry and fluorescent staining techniques. As these values were obtained alongside the complete blood count, the results were more accurate than manual counts and did not incur any additional costs. The number and percentage of NRBCs were recorded at the time of admission to the ICU, as well as on the day of discharge from the ICU and at 24 and 48 hours before discharge from the ICU. Hemoglobin, neutrophil, white blood cell, platelet, lymphocyte, CRP, procalcitonin, creatinine, and lactate values were obtained at admission from the hospital information management system. The neutrophil/lymphocyte ratio was calculated. SOFA and APACHE II scores calculated within the first 24 hours of admission to the ICU were recorded.

Scoring systems were also evaluated daily and recorded in the hospital information management system. Patients were categorized into 2 groups: those who lived (survivor group) and those who died (mortality group).

STATISTICAL ANALYSIS:

The normality of continuous variables was evaluated using the Kolmogorov-Smirnov test. Non-normally distributed continuous variables were presented as median values, with minimum and maximum (range) values provided, and analyzed using the Mann-Whitney U test. Continuous variables that followed a normal distribution are expressed as mean±standard deviation (SD) values and were analyzed. Box plots and bar charts were used to show the distribution of the data. Categorical variables are presented as proportions. The chi-square test was used to analyze categorical variables. The best cut-off values for continuous variables exhibiting significant differences in paired comparisons were found using receiver operating characteristic (ROC) curves. Calculations were made to determine metrics, including sensitivity, specificity, the area under the ROC curve (AUC), positive likelihood ratio (PLR), and negative likelihood ratio (NLR). Statistical significance was defined as a P value of less than 0.05. Python 3.11.6 and IBM SPSS Statistics, version 26.0 (IBM Corp, Armonk, NY, USA) were used for data analysis, along with the libraries “seaborn”, “scikit-learn”, “matplotlib”, and “statsmodels”.

Results

A total of 160 patients with sepsis were included in the study. The median age was 79 (range: 29–99) years; 46.25% of the patients were male. When evaluating the etiology of sepsis, 60% of infections were from the respiratory system, 28.75% from the urinary system, 6.25% from soft tissue or skin, 3.75% from the gastrointestinal system, and 1.25% were catheter-related. A total of 81 (50.625%) patients died. Demographic characteristics, diagnostic groups, and clinical outcomes of the patients are summarized in Table 1.

The median scores for clinical assessment scales were as follows: APACHE II, 22 (range: 8–41); SOFA, 6 (range: 2–13); and GCS, 14 (range: 5–15). The median value for ICU length of stay was 8 days (range: 3–84 days) and for total hospitalization, 17.5 days (range: 3–258 days).

Of the 79 patients in the survivor group, 33 (41.8%) were male, with a median age of 80 years (range: 29–99 years). The median ICU stay was 6 days (range: 3–62), and the median total hospitalization was 18 days (range: 5–258). Among the 81 patients in the mortality group, 41 (50.6%) were male, and the median age was 78 years (range: 41–98). In this group, the median ICU stay was 10 days (range: 3–84), and the median total hospitalization was 15 days (range: 3–105). The clinical and laboratory results of the groups are shown in Table 2.

While the SOFA and APACHE II scores were higher in the mortality group, the GCS was lower (P<0.001 for all). In laboratory test results, the neutrophil-to-lymphocyte ratio, lactate, and procalcitonin values were higher in the mortality group (P=0.032, P<0.001, P=0.048, respectively). ICU hospitalization days were higher in the mortality group (P=0.033). When the groups were compared for NRBC count and NRBC percentage, although the NRBC count and percentage 24 hours before discharge were higher in the mortality group, there was no statistically significant difference between the 2 groups. NRBC counts at ICU admission and at discharge were higher in the mortality group, reaching statistical significance. The NRBC count at admission was P=0.029, and at discharge P=0.004. Similarly, the NRBC percentage was significantly higher in the mortality group, with P=0.038 at admission and P=0.009 at discharge. In addition, the NRBC count measured 48 hours before discharge was higher in the mortality group (P=0.02).

Analysis of median values showed the NRBC at admission was 0/μL in the survivor group and 10/μL in the mortality group. While the highest value measured in the survivor group was 160/μL, this value increased up to 1550/μL in the mortality group. In addition, the highest values were found in NRBC values measured at discharge and 24 hours before discharge. In the survivor group, the maximum value at the time of discharge was 640/μL, while the maximum value 24 hours before discharge was 180/μL. The maximum values were notably higher in the mortality group, with maximum values measuring 10130/μL at discharge and 10520/μL 24 hours before discharge. Box plots and bar charts for NRBC values are shown in Figure 1.

ROC curve analyses showed the highest value was observed for the NRBC value at discharge, with a value of 0.631; the cut-off value was 10 NRBC/uL. The sensitivity was 0.63, and the specificity was 0.582. The AUC of the NRBC percentage at discharge was 0.621, and the AUC of the NRBC count at admission was 0.6. The cut-off value for NRBC at admission was 20/μl with a specificity of 0.848. This was also the highest value, including scoring within the evaluation. The highest AUC value in the analyses was associated with GCS. The cut-off value for GCS was 13, and the AUC was 0.788, with sensitivity of 0.7 and specificity of 0.77. APACHE II and SOFA scores showed better performance than NRBC values, with an AUC of 0.77, and cut-off values of 21 and 6, respectively. Laboratory values and AUCs of the scoring are shown in Table 3. ROC curves are shown in Figure 2.

Discussion

LIMITATIONS:

The single-center, retrospective design of our study may introduce bias into the results. The effectiveness of NRBC should be confirmed by further studies. Our analysis included sepsis groups with different etiologies and subgroups, rather than a single sepsis subgroup. Additional diseases and comorbid conditions beyond the sepsis diagnosis were not considered. Blood product transfusions administered during ICU admission were not taken into account, which may have affected the NRBC count. Finally, NRBC count and percentage were analyzed at admission, discharge, and 24 and 48 hours before discharge, whereas other markers and scoring systems were evaluated only at admission.

Conclusions

Regular monitoring of NRBC count and percentage, rather than relying on a single measurement, and examining their trends may play an important role in patient follow-up and treatment. Our study highlighted that NRBC is beneficial in assessing the clinical severity and course of sepsis patients monitored in the ICU, emphasizing the need for further research to determine threshold values and evaluate the optimal evaluation period. Consideration of NRBC with routine parameters and scoring systems may contribute to early diagnosis and treatment. This may improve the outcome of patients with sepsis in the ICU. Future studies involving larger, more homogeneous, and well-defined populations will be both enlightening and informative.