DOI: https://doi.org/10.21203/rs.3.rs-2296446/v1
Background: Systemic inflammation and immune system dysfunction play important roles in the pathophysiology of cirrhosis. The neutrophil-to-lymphocyte ratio (NLR) is a marker associated with systemic inflammation and immune dysregulation in cirrhotic patients. NLR is inexpensive and may predict decompensation and mortality in these patients. In this study, we aimed to investigate the association between NLR and the presence of ascites as a complication in cirrhosis.
Methods: A retrospective observational cross-sectional study was conducted in 61 adult cirrhotic patients without documented malignancy, systemic infection, and autoimmune diseases at our hospital between January 2015 to December 2018. NLR, presence of ascites, and all other variables were collected from the first day of presentation.
Results: There were 31 patients with ascites and 30 without ascites. The mean NLRs were 2.72±1.20 and 3.53±1.51 in patients with and without ascites (p=0,022). ROC curve analysis demonstrated a NLR cut-off value of 2.70 (AUC 0.660, p=0.032). Cirrhotic patients with NLR <2.70 is 4.354 times more likely to have the presence of ascites (95% CI: 1,471-12,885, p=0.006).
Conclusion: Lower neutrophil-to-lymphocyte ratio is associated with the presence of ascites in cirrhosis. Patients with NLR of <2.70 are four times more likely to have the presence of ascites. This finding supports the evidence of neutropenia in decompensated cirrhosis. Further studies are required to elucidate the prognostic utility of NLR in cirrhosis.
Cirrhosis is the end-stage of progressive liver fibrosis characterized by the formation of regenerative nodules surrounded by fibrous septa as a response to chronic liver injury, which subsequently leads to portal hypertension and end-stage liver disease [1]. Cirrhosis is a leading cause of morbidity and mortality globally. In 2017, cirrhosis caused more than 1.32 million deaths, constituting 2.4% of total deaths globally. There were 10.6 million prevalent cases of decompensated cirrhosis and 112 million prevalent cases of compensated cirrhosis globally in 2017 [2].
Ascites, the pathologic accumulation of fluid in the peritoneal cavity, is the most common complication of cirrhosis and develops in about 50% of patients in ten years after initial diagnosis.[3] The development of ascites in cirrhosis is associated with a poor prognosis and impaired quality of life. The 1-year mortality risk increases from 1–3% to 15–20% after the development of ascites and as high as 50% in 5 years [4, 5].
Neutrophil-to-lymphocyte ratio (NLR), the ratio between neutrophil count and lymphocyte count in peripheral blood, is a systemic inflammation marker representing the degree of inflammation and the relationship between two different immune pathways. Neutrophil count reflects the ongoing process of inflammation, whereas lymphocyte count represents the immune regulation pathway. NLR is an expensive biomarker and easily calculated from a routinely available complete blood count. NLR has been consistently associated with poor outcomes and mortality in cirrhosis [6, 7].
Systemic inflammation and immune system dysfunction play important roles in the pathophysiology of cirrhosis. Portal hypertension causes intestinal bacterial translocation and thus leads to increased inflammation. On the other hand, it also contributes to the increase in capillary hydrostatic pressure, resulting in the development of ascites as a complication [8]. In this study, we aim to investigate the association between NLR and the presence of ascites in cirrhosis.
We performed a retrospective observational cross-sectional study in cirrhotic patients aged 18 to 75 years. Subjects were obtained from medical record data of cirrhotic patients at Dr. Sardjito General Hospital, Yogyakarta, Indonesia during 2015–2018, who meet the inclusion and exclusion criteria. Patients were included if the diagnosis of cirrhosis was clearly considered on the basis of on clinical and radiological features. Patients with documented malignancy, systemic infection, sepsis, and autoimmune diseases were excluded from the study.
Data extraction included patient demographics, NLR, ascites, cirrhosis etiology, serum albumin, and the presence of type 2 diabetes mellitus – all of which were collected from the day of first presentation. NLR was measured as the ratio between neutrophil and lymphocyte percentage obtained from peripheral blood laboratory examination. The presence of ascites was diagnosed with ultrasonography and recorded as ‘with ascites’ or ‘without ascites’. We also evaluated a few confounding variables, including age, etiology, serum albumin, type 2 diabetes mellitus.
Patient characteristics were described using mean ± standard deviation (SD), or frequency and percentage, as appropriate. Normality of data distribution was determined with Saphiro Wilk test. Mean difference of NLR between patients with ascites and without ascites was tested with independent t test or Mann-Whitney test. Chi square or Fisher exact test was used to determine whether NLR can predict the presence of ascites. Cut-off point for NLR was obtained from Receiver Operating Characteristic (ROC) curve analysis. Results were considered statistically significant if p value was < 0.05. Statistical analysis was carried out with IBM SPSS 24.
This study enrolled a total of 61 subjects, consisting of 39 (63,9%) males and 22 (36,1%) females, who met the inclusion and exclusion criteria of this study. The mean age of the patients was 54,56 ± 10,71 years, ranging from 20 to 75 years old. Thirty-one (50,8%) subjects presented with ascites and 30 (49,2%) subjects without ascites. Baseline characteristics of subjects are demonstrated in Table 1. None of the confounding variables significantly influenced the presence of ascites (Table 2).
|
Variable |
Mean ± SD |
Median (minimum-maximum) |
Frequency |
|---|---|---|---|
|
Age (years) |
54.56 ± 10.71 |
56 (20–75) |
|
|
Sex |
|||
|
- Male n(%) |
39 (63.9%) |
||
|
- Female n(%) |
22 (36.1%) |
||
|
Ascites |
|||
|
- With ascites n(%) |
31 (50.8%) |
||
|
- Without ascites n(%) |
30 (49.2%) |
||
|
CPT |
|||
|
- Class A |
26 (42.6%) |
||
|
- Class B |
29 (47.5%) |
||
|
- Class C |
6 (9.8%) |
||
|
Etiology |
|||
|
- Hepatitis B n(%) |
39 (63.9%) |
||
|
- Hepatitis C n(%) |
6 (9.8%) |
||
|
- Not Hepatitis B/C n(%) |
12 (19.7%) |
||
|
- Unknown n(%) |
4 (6.6%) |
||
|
Albumin (mg/dL) |
3.25 ± 0.80 |
3.23 (1.50–4.88) |
|
|
Type 2 DM |
|||
|
- Present |
10 (16.4%) |
||
|
- Not present |
51 (83.6%) |
|
Variable |
With ascites (n = 31) |
Without ascites (n = 30) |
p value* |
|---|---|---|---|
|
Mean ± SD/Frequency |
Mean ± SD/Frequency |
||
|
Age (years) |
54.64 ± 8.00 |
54.47 ± 13.08 |
0.949 |
|
Etiology |
0.581 |
||
|
- Hepatitis B |
22 |
17 |
|
|
- Hepatitis C |
3 |
3 |
|
|
- Not Hepatitis B/C |
4 |
8 |
|
|
- Unknown |
2 |
2 |
|
|
Albumin (mg/dL) |
3.40 ± 0.82 |
3.11 ± 0.76 |
0.157 |
|
DM |
0.731 |
||
|
- With DM |
6 |
4 |
|
|
- Without DM |
25 |
26 |
|
|
Neutrophil percentage (%) |
58.93 ± 13.12 |
66.24 ± 8.53 |
0.024 |
|
Lymphocyte percentage (%) |
25.38 ± 8.02 |
21.42 ± 7.18 |
0.036 |
|
Neutrophil-to-lymphocyte ratio |
2.72 ± 1.20 |
3.53 ± 1.51 |
0.022 |
| *Independent t test, statistically significant if p < 0.05 | |||
Mean NLR is significantly lower in patients with ascites compared to those without ascites (2.72 ± 1.20 vs 3.53 ± 1.51; 95% CI:-1.51 to -0.12; p = 0.22) (Fig. 1 and Table 2). An additional analysis to test the mean difference of each NLR component (neutrophil count and lymphocyte count) was also conducted. Neutrophil count and lymphocyte count, respectively, was significantly lower and higher in ascites group compared to non-ascites group (p = 0.024; and p = 0.036; Table 2).
Cut-off of NLR, determined with ROC curve analysis, was 2.70 (AUC = 0,660; p = 0,032; Sensitivity: 73.3%; Specificity: 61.3%) (Fig. 2). Cirrhotic patients with NLR of < 2.70 is 4.354 times more likely to have the presence of ascites (95% CI: 1,471 − 12,885, p = 0.006) (Table 3).
|
Variable |
Ascites |
p value* |
Odds Ratio |
95% CI |
|
|
With ascites |
Without ascites |
||||
|
NLR < 2.70 |
19 |
8 |
0,006 |
4.354 |
1.471–12.885 |
|
NLR > 2.70 |
12 |
22 |
|||
| *Chi-square; CI confidence interval, statistically significant if p < 0.05 | |||||
Our study examined the association between NLR and the presence of ascites in cirrhosis. Our results demonstrate a significant difference of mean NLR between cirrhotic patients with ascites and without ascites. NLR is significantly lower in patients with ascites compared to those without ascites. Mean neutrophil count and lymphocyte count are also significantly different between the two groups, with relative neutropenia and relative lymphocytosis exhibited in patients with ascites. Cirrhotic patients with NLR of < 2.70 is 4.354 times more likely to have the presence of ascites, with a 73.3% sensitivity and 61.3% specificity.
The results of our study are somewhat contradictory to the findings demonstrated in numerous previous studies about the association between high NLR with worse outcomes or high mortality in cirrhosis. Biyik et al. showed that NLR is a predictor of mortality independent of Child Turcotte Pugh (CTP) and Model for End-Stage Liver Disease (MELD) scores in cirrhotic patients [9]. Kalra et al. also reported that high NLR may aid in determining risk for cirrhotic decompensation [10].
Patients with decompensated cirrhosis is prone to develop neutropenia. Some hypotheses have been suggested to explain this phenomenon. The most common hypothesis is splenomegaly and hypersplenism, which causes increased clearance of polymorphonuclear (PMN) cells and the presence of hematopoietic progenitor cell inhibitor in serum [11]. However, this concept has never been proven since neutropenia was shown to persist even after splenic embolization or splenectomy [12].
Neutrophil is continuously produced in bone marrow and therefore, a similar number of PMN cells must die to maintain the cellular homeostasis. Apoptosis or programmed cell death is a process that regulates the lifespan of inflammatory cells. Therefore, apoptosis rate affects neutrophil count in the blood. In decompensated cirrhosis, an increased rate of apoptosis is found. There are at least two mechanisms explaining this event. First, recruitment of TNF-R and Fas receptors induce apoptosis by neutrophil or phagocytosis of neutrophils that have been opsonized by IgG and complement. Second, there is an increase in production of reactive oxygen species (ROS) in neutrophils of patients with decompensated cirrhosis and ROS is also one of the apoptotic signals. Both these mechanisms lead to activation of caspase enzymes, which are main executors of apoptosis. Apoptosis is triggered by the binding of Fas receptor and TNF-R in extensive pathway of apoptosis and involves caspase-8, 10, or 3. PMN cells in cirrhotic patients with ascites have higher caspase-3 activity than those without ascites. These findings also contribute to explain the phenomenon of neutropenia in cirrhotic patients [13]. However, until recently, explanation on neutropenia in decompensated cirrhosis has not been satisfying and therefore requires further studies.
Meanwhile, we did not find any literature or studies that explain the phenomenon of relative lymphocytosis in our study. We speculate that the relative lymphocytosis in ascites group is due to the associated relative neutropenia. Overall, due to neutropenia in decompensated cirrhosis, NLR in patients with ascites tends to be lower than those without ascites.
This study has several limitations. Some factors confounding the presence of ascites could not be controlled or analyzed, such as patients’ treatment and diet, especially sodium intake. Our study did not classify the severity of ascites and type of patient care (inpatient or outpatient). We also did not analyze the absolute neutrophil and lymphocyte count due to unavailability of retrospective data. Moreover, this study utilized a cross-sectional approach which was not able to represent the full course of the disease since exposure and outcome were measured concurrently. Further studies on the relationship between NLR and the presence of ascites need to be conducted by considering more confounding variables, using the cohort design to represent the full disease course, and addressing other limitations of this study.
Lower NLR is associated with the presence of ascites in cirrhosis. Patients with NLR of < 2.70 are four times more likely to have the presence of ascites. This finding supports the evidence of neutropenia in decompensated cirrhosis. Further studies are required to elucidate the prognostic utility of NLR in cirrhosis.
Ethics approval and consent to participate
Subjects have been informed and have provided consent for their data to be included in this study. This study was conducted according to the World Medical Association Declaration of Helsinki and has been approved by the Medical and Health Research Ethics Committee (MHREC) of Faculty of Medicine, Universitas Gadjah Mada – Dr. Sardjito General Hospital (No. KE/FK/0353/EC/2018).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
The authors declare that they have not received any funding for the research.
Authors’ contributions
WW analyzed and interpreted the patient data and was a major contributor in writing the manuscript. NR, CT, and FI supervised the research, validated the results and interpretation, and reviewed the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Not applicable.