Hepatic Steatosis Is Associated with High White Blood Cell and Platelet Counts

The incidence of hepatic steatosis is increasing globally, and it is important to identify those at risk to prevent comorbidities. Complete blood count is a simple, convenient, and inexpensive laboratory examination which can be used to obtain white blood cell (WBC) and platelet counts. The aims of this study were to investigate the relationships between WBC and platelet counts with hepatic steatosis, and whether WBC and platelet counts were associated with the severity of hepatic steatosis. We enrolled 1969 participants residing in southern Taiwan who took part in a health survey from June 2016 to September 2018 in this cross-sectional study. None of the participants were heavy alcohol users or had a history of hepatitis B or C. We collected laboratory data, and the severity of hepatic steatosis was determined by abdominal ultrasound. The overall prevalence rate of hepatic steatosis was 42.0%. There were significant trends of stepwise increases in WBC count (p < 0.001) corresponding to the severity of hepatic steatosis. After multivariable linear regression analysis, hepatic steatosis was significantly associated with high WBC count (coefficient β, 0.209; 95% confidence interval (CI), 0.055 to 0.364; p = 0.008) and high platelet count (coefficient β, 12.213; 95% CI, 6.092 to 18.334; p < 0.001); also, higher WBC counts corresponded with the severity of hepatic steatosis.


Introduction
The global prevalence of nonalcoholic fatty liver disease (NAFLD) is around 25% [1]; it has rapidly increased in Asia over the last decade from 25% to 34% [2]. NAFLD is a known risk factor for the development of hepatocellular carcinoma, advanced liver disease, and related complications, and it has also been strongly associated with cardiovascular diseases, metabolic syndrome, diabetes, and extrahepatic malignancy [3][4][5]. The pathogenesis of NAFLD is not fully understood, but insulin resistance and chronic inflammation have been shown to play vital roles [6]. Insulin resistance leads to abnormal triglyceride accumulation in hepatocytes, causing hepatic steatosis and then inflammation mechanisms including lipotoxicity and immune reaction, leading to nonalcoholic steatohepatitis (NASH) and activating hepatic stellate cells to cause hepatic fibrosis [7,8]. Accordingly, patients with NAFLD have been reported to have elevated levels of inflammatory biomarkers, including high-sensitivity C-reactive protein (CRP) [9], tumor necrosis factor-α (TNF-α) [10], and interleukin-6 (IL-6) [11]. Moreover, this chronic inflammation further worsens insulin

Participant Recruitment
This cross-sectional study was conducted in southern Taiwan from June 2016 to September 2018. The study protocol was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (number: KMUHIRB-G(II)-20190011), and all participants gave informed consent before joining the study.
The participants were recruited from a health survey which was promoted through advertisements. They all underwent physical examinations, and an experienced physician recorded their clinical histories. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were also measured. All of the participants also underwent a face-to-face interview with a researcher, during which they completed a questionnaire asking about alcohol history. For men, heavy drinking is typically defined as consuming 15 drinks or more per week in his lifetime. For women, heavy drinking is typically defined as consuming 8 drinks or more per week in her lifetime. Participants who did not meet the above criteria and were not social drinkers were defined as having an alcohol history. Heavy alcohol users and those with a history of hepatitis B or C were excluded. In addition, hemochromatosis, Wilson disease, and autoimmune hepatic disorders were excluded. A total of 2446 participants with complete data were screened, of whom 477 were excluded due to a history of severe alcohol consumption (n = 49), hepatitis B (n = 307), hepatitis C (n = 102), and hepatitis B and C (n = 19). The remaining 1969 participants were analyzed ( Figure 1). drinks or more per week in her lifetime. Participants who did not meet the above criteria and were not social drinkers were defined as having an alcohol history. Heavy alcohol users and those with a history of hepatitis B or C were excluded. In addition, hemochromatosis, Wilson disease, and autoimmune hepatic disorders were excluded. A total of 2446 participants with complete data were screened, of whom 477 were excluded due to a history of severe alcohol consumption (n = 49), hepatitis B (n = 307), hepatitis C (n = 102), and hepatitis B and C (n = 19). The remaining 1969 participants were analyzed ( Figure 1).

Figure 1.
Flowchart of study population.

Collection of Demographic, Medical, and Laboratory Data
Baseline data on lifestyle habits (smoking and alcohol consumption), medical history (diabetes mellitus (DM) and hypertension), and demographics (age and sex) were recorded. In addition, the following laboratory data were also recorded at baseline: WBC count, hemoglobin, platelet count, fasting glucose, triglycerides, total cholesterol, highdensity lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), estimated glomerular filtration rate (eGFR), and uric acid. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI eGFR) [45].

Assessment of Hepatic Steatosis
Hepatic steatosis was defined as the presence of hepatic steatosis on liver ultrasonography that could not be explained by the secondary accumulation of hepatic fat or acute/chronic liver diseases, such as treatment with steatogenic drugs and severe alcohol use [46,47]. Liver ultrasonography was performed by experienced radiologists who were blinded to the participants' biochemical data and clinical diagnoses. Hepatic steatosis was defined as the presence of at least two of the following: greater echogenicity in the liver compared to the kidneys, gradual attenuation of far-field ultrasound echo, vascular blurring, and diffuse increase in near-field ultrasound echo ('bright liver') [47,48]. Steatosis is

Collection of Demographic, Medical, and Laboratory Data
Baseline data on lifestyle habits (smoking and alcohol consumption), medical history (diabetes mellitus (DM) and hypertension), and demographics (age and sex) were recorded. In addition, the following laboratory data were also recorded at baseline: WBC count, hemoglobin, platelet count, fasting glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), estimated glomerular filtration rate (eGFR), and uric acid. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI eGFR) [45].

Assessment of Hepatic Steatosis
Hepatic steatosis was defined as the presence of hepatic steatosis on liver ultrasonography that could not be explained by the secondary accumulation of hepatic fat or acute/chronic liver diseases, such as treatment with steatogenic drugs and severe alcohol use [46,47]. Liver ultrasonography was performed by experienced radiologists who were blinded to the participants' biochemical data and clinical diagnoses. Hepatic steatosis was defined as the presence of at least two of the following: greater echogenicity in the liver compared to the kidneys, gradual attenuation of far-field ultrasound echo, vascular blurring, and diffuse increase in near-field ultrasound echo ('bright liver') [47,48]. Steatosis is graded as follows: absent (score 0), when the echotexture of the liver is normal; mild (score 1), when there is a slight and diffuse increase of liver echogenicity with normal visualization of the diaphragm and of the portal vein wall; moderate (score 2), in the case of moderate increase in liver echogenicity, with slightly impaired appearance of the portal vein wall and the diaphragm; severe (score 3), in the case of marked increase in liver echogenicity with poor or no visualization of portal vein wall, diaphragm, and posterior part of the right liver lobe [49].

Statistical Analysis
Continuous variables are presented as mean ± standard deviation or median (25-75th percentile), and categorical data are presented as number and percentage. Differences between categorical variables were compared using the chi-squared test, and differences between continuous variables were compared using the independent t test. Multiple comparisons among hepatic steatosis severity groups were performed using one-way analysis of variance followed by a Bonferroni-adjusted post hoc test. Spearman correlation was performed to identify the association between WBC and platelet counts with hepatic steatosis severity groups. Linear regression analysis was used to identify associations between WBC count and platelet count with hepatic steatosis. Significant variables in univariable analysis were entered into multivariable analysis. p-values < 0.05 were considered to be statistically significant. SPSS version 19.0 for Windows was used for all statistical analyses (SPSS Inc. Chicago, IL, USA).

Discussion
NAFLD is a spectrum of liver disease, from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH) with progressive hepatic inflammation and a higher risk of liver fibrosis and hepatic comorbidities. Cardiovascular events and extra-hepatic malignancies are the first and second most common causes of mortality in patients with NAFLD [13]. It is important to identify the presence and severity of NAFLD as early as possible to help prevent comorbidities. In this study, we observed that hepatic steatosis was associated with high WBC and platelet counts, and higher WBC counts corresponded with the severity of hepatic steatosis in 1969 participants residing in southern Taiwan.
The first important finding of this study is that the participants with hepatic steatosis had a higher WBC count. Furthermore, there was a significant trend of a stepwise increase in WBC count corresponding to the severity of hepatic steatosis. Lee et al. reported a positive correlation between WBC count and the prevalence of NAFLD, also showing an increased risk of NAFLD according to higher WBC count [36]. Yu et al. revealed that a higher WBC count is a risk factor of NAFLD, and that the stepwise increase in WBC count is a positive correlation with the risk of NAFLD combined with H pylori infection [38]. Further cohort studies also confirmed the association between an elevated WBC count and the risk of NAFLD [22,37]. In addition, Gokulakrishnan et al. reported an association between the severity of NAFLD and WBC count in south Indians in 2012 [50], which is consistent with our study result. Several mechanisms explain increased WBC count in hepatic steatosis [51]. First, insulin resistance causes hyperinsulinemia, leading to de novo hepatic lipogenesis and steatohepatitis [23]. The excess fatty acid then results in lipotoxic lipids and oxidative stress in hepatocytes, leading to liver necrosis. The apoptosis of hepatocytes produces damageassociated molecular patterns (DAMPs) and activates inflammatory signals, which then increases WBC count [24]. Second, interacting with the microbiome in the intestinal system and liver disease, so-called gut-liver axis has been established [3]. In health conditions, the permeability of the intestine is well controlled to avoid pathogen-associated molecular patterns-such as bacteria and bacterial toxic products including lipopolysaccharides-and endotoxin exposure via the hepatic portal system [52]. However, alcohol intake and a highfat diet will cause dysbiosis [53], which leads to intestinal epithelial loosening and initiates intestinal inflammation by releasing inflammatory cytokines-such as interferon-gamma and interferon-alpha-through mucosal immune system cells [54]. The cellular toxic metabolites passing through the hyperpermeable intestinal barrier elicits liver inflammation and subsequently causes liver disease [54]. Third, as well as being an energy-storing organ, adipose tissue is the largest endocrine organ producing adiponectin, leptin, TNF-α, and IL-6, which leads to systemic inflammation and insulin resistance, thereby further exacerbating NAFLD [55]. In addition, a previous animal model showed that adipose death could trigger liver injury and inflammation [56]. Progressive inflammation plays an important role in the pathogenesis of the progression of NAFLD [57]. In addition, an increase in hs-CRP has been associated with the severity of NAFLD [58]. WBC count is also a surrogate marker of chronic inflammation, and it corresponded with the severity of hepatic steatosis in our study.
The second important finding of this study is the higher platelet count in the participants with hepatic steatosis. The association between platelet count and NAFLD is controversial. Several studies have observed a negative correlation between platelet count and the severity of NAFLD [39][40][41]. Possible mechanisms include liver cirrhosis with splenomegaly and decreased thrombopoietin production in patients with advanced liver disease [40]. However, Sung et al. reported a positive association between platelet count and the incidence of NAFLD [42]. In addition, Afagh et al. found that a higher platelet count was associated with more severe NAFLD [43], and the platelet count in patients with NASH has been reported to be higher than in patients without NASH [44]. Platelets play a role in liver regeneration after liver damage; however, they can also trigger the pathogenesis of liver fibrosis [59] by producing transforming growth factor-beta and platelet-derived growth factor, which activate fibrocompetent cells, notably hepatic stellate cells [57]. The fibrocompetent cells then remodel extracellular matrix components, leading to fibrosis [60]. In addition, platelets have been shown to induce innate and adaptive immune responses, thereby causing progressive liver damage in patients with chronic viral hepatitis and NAFLD [61]. Previous studies have also demonstrated an association between an elevated platelet count with metabolic syndrome [62] and insulin resistance [31]. Metabolic syndrome causes systemic chronic inflammation and an increase in IL-6, which are also associated with increased platelet production, and more risk factors for metabolic syndrome have been positively associated with a higher platelet count [62]. Insulin resistance has also been shown to increase platelet count through an increase in thrombopoietin production by excess adipose tissue [63]. The various mechanisms associated with platelets may have different effects on platelet count, and may be related to disease stage [59,60].
Another important finding of this study is that a high uric acid level was associated with high WBC and platelet counts. Hyperuricemia is defined as high levels of uric acid, an end-product of purine metabolism [64]. A diet rich in purine and alcohol consumption are major risk factors for hyperuricemia, and genetic factors are also important [64]. Uric acid produces free radicals, particularly fatty acids, which cause systemic inflammation and vascular injury [65], and subsequently cardiovascular disease, metabolic syndrome, chronic kidney disease, and insulin resistance [66]. Hyperinsulinemia has been shown to cause decreased uric acid secretion in renal tubules [67]. Two meta-analyses found that higher levels of uric acid were associated with the prevalence of NAFLD [68,69]. Moreover, a linear relationship between uric acid and the incidence of NAFLD has also been reported [70]. Furthermore, an observational study reported a higher uric acid level in non-obese patients with NAFLD compared with obese patients [71]. Hyperuricemia is accompanied by inflammatory markers, including IL-6, IL-18, hs-CRP, and TNF-α [72]. Su et al. reported a significant association between uric acid concentration and WBC count, but no significant association between uric acid and platelet count [73]. In addition, Liu et al. reported that hyperuricemia was associated with a higher WBC count, and that this association was independent of chronic kidney disease [74]. In contrast to these studies, Tayefi et al. reported positive correlations between platelet count and mean platelet volume with serum uric acid in newly diagnosed hypertensive patients [75].
There are several limitations to this study. First, we could not ascertain causal relationships or long-term clinical outcomes due to the cross-sectional design of this study. Prospective studies with NAFLD and immune function assessments and a longer follow-up period are needed to verify our results. Second, we did not evaluate sleeping or economic status in our analysis, both of which may also be associated with the development of NAFLD. Third, all of the participants in this study were Taiwanese, which may limit the generalizability of our results to other populations. Fourth, only participants who were willing to attend the study were included, making it considerably more difficult to interpret standard errors and confidence intervals. Fifth, insulin resistance is a hallmark of NAFLD and it plays a pivotal role in the pathogenesis of the disease. However, insulin level was not measured in this study. Therefore, we could not evaluate the association between insulin resistance and NAFLD. Sixth, we assessed the severity of NAFLD according to ultrasound findings. However, ultrasound of the liver is qualitative and may not be sufficient for scoring. In addition, future studies should include data on differentiated WBC count to further investigate impaired innate and acquired immune systems. Finally, the values of WBC and platelets are within the normal range in each group of participants. Therefore, it would be difficult to consider the measurement of WBC or platelet count helpful in the diagnosis of hepatic steatosis.
In conclusion, we found hepatic steatosis was associated with high WBC and plate-let counts, and higher WBC counts corresponded with the severity of hepatic steatosis; also, the pathogenesis may have been related to chronic inflammation. We suggest that WBC count and platelet count may play a role in stratifying hepatic steatosis.