The Association between Platelet count and Liver volume after eradication of Hepatitis C virus by Direct-acting Antivirals: a retrospective study

Background: Although most patients who obtain sustained virological response (SVR) show improved liver function, some patients show decreased platelet counts after eradication of hepatitis C virus (HCV). The aim of this retrospective study was to clarify the association of liver and spleen volumes with platelet count after SVR achieved by direct-acting antiviral (DAA) treatment. Methods: This study enrolled 36 consecutive patients treated by DAAs who obtained SVR between September 2014 and December 2018. Liver and spleen volumes were derived from computed tomography scans obtained at pretreatment, SVR, and 48 weeks after SVR. No patient developed hepatocellular carcinoma during this study. Results: Compared with pretreatment, the median AST, ALT, albumin serum levels, and platelet counts improved significantly at SVR and 48 weeks after SVR. The liver/spleen volumes per body weight were decreased significantly from 22.5/4.2 mL/kg to 21.1/3.6 mL/kg at 48 weeks after SVR. The change in liver volume was associated with change in platelet count, and the change in spleen volume was negatively associated with change in serum albumin level. Multivariate analysis identified change in liver volume (≥95%, odds ratio 76.9, P = 0.005) as the factor associated with improvement in the platelet count at 48 weeks after SVR. The patients with increased liver volume at 48 weeks after SVR showed increased platelet count. Conclusions: Both liver and spleen volume once decreased significantly by eradication of HCV. The patients with re-increased liver volume showed rapid increase of platelet count. Results are presented as n (%) for qualitative data or as median (range) for quantitative data. Results are presented as n (%) for qualitative data or as median (range) for quantitative data.


Introduction
Hepatitis C virus (HCV) infection is one of the most important public health concerns in the world. An estimated 270-300 million people are infected worldwide, and the prevalence of the infection is expected to peak in the next 10-20 years [1]. The proportion of patients obtaining sustained virological response (SVR) due to directacting antiviral (DAA) treatment subsequently dramatically increased to more than 95% [2].
Most patients treated by DAAs show improved liver function after obtaining SVR.
However, some patients have persistently abnormal liver function test results after SVR. Platelet counts also improved after SVR, and the change in platelet counts was greater in patients obtaining SVR than patients without SVR after IFN-based therapy [3]. Thrombocytopenia has been associated with Hepatocellular carcinoma (HCC) development in cirrhotic patients with HCV infection [4]. On the other hand, several studies showed that thrombocytosis is associated with bigger tumor size, more frequent metastasis and poor prognosis [5][6][7] Though it is unclear if thrombocytopenia or thrombocytosis per se is a risk factor for HCC development, evaluating the predictive factor of platelet count after SVR is important.
Liver and spleen volumes derived from computed tomography (CT) scans have been used to assess graft volumes, and to predict operative outcomes in patients with decompensated liver disease [8,9]. Several study reports have discussed the relationship between liver volume and functional reserve in patients with cirrhosis [10][11][12]. In cirrhotic patients, liver volume decreased in accordance with progression of hepatic fibrosis, and liver and spleen volumes might be related to the degree of portal hypertension [13][14][15]. However, the association between remodeling changes in liver/spleen volume and changes in platelet count in patients undergoing DAA therapy has remained unclarified.
The aims of this study were to assess changes in liver/spleen volumes and platelet counts over a 2-year period in patients who obtained SVR by DAA therapy, and to identify the predictors of increased platelet counts.

Patients
We performed a retrospective single-center study of consecutive patients with HCV infections from the outpatient clinic at the Department of Gastroenterology and

Statistical analysis
The liver volume per body weight (LV/BW) and spleen volume per body weight (SV/BW) were calculated at each point. The change in biological and imaging parameters at 48 weeks after SVR from those at baseline were shown as delta (%).
Distributions of the characteristics of the study patients were assessed by the chisquared test or Mann-Whitney U test, as appropriate. Pearson coefficient were performed to analyze the factors associated with change in liver and spleen volume.
In multivariate logistic regression analysis, platelet count, albumin, total bilirubin, and change in LV/BW at baseline to 48 weeks after SVR were included as confounders. Odds ratios (ORs) and 95% confidence intervals (CIs) were also calculated. Statistical comparisons were performed by SPSS software (SPSS Inc., Chicago, IL). All P-values less than 0.05 by the two-tailed test were considered significant. Table 1  Factors associated with improvements in platelet counts The median platelet counts increased significantly from 10.3 x 10 4 /μL at baseline to 11.6 x 10 4 /μL at 48 weeks after SVR. Among the study population, 10 (27.8%) patients showed a platelet count that decreased from baseline to 48 weeks after SVR. Table 2 lists the characteristics of study patients stratified by changes in their platelet counts. The differences between gender, age, BMI, alcohol intake, and the prevalence of complications between the patients whose counts increased and those whose counts decreased were not significant. The baseline liver function tests of the 2 groups also did not reflect the changes in platelet counts. The median LV/BW values at baseline of the patients with decreased platelet count vs those with increased platelet count were 24.0 mL/kg and 21.1 mL/kg, respectively (P = 0.037).

Patient characteristics
The difference between median SV/BW values at baseline in the patients with decreased platelet count vs those with increased platelet count was not significant.
The change in median LV/BW value of patients with decreased platelet counts between baseline to 48 weeks after SVR (87%) was significantly smaller than the change in median value of patients with increased platelet count (97%) (P < 0.001).
Differences between the changes in SV/BW values were not significant.
To identify factors significantly associated with improved platelet counts, we performed a multivariate logistic regression analysis. The following factors were evaluated by multivariate analysis: platelet count, albumin, total bilirubin, change in LV/BW at baseline to 48 weeks after SVR. The change in LV/BW at 48 weeks after SVR (≥95%) was identified to be an independent factor associated with improved platelet count (adjusted OR 76.9, 95% CI 3.78-1000; P = 0.005) ( Table 3).
Association between liver volume and platelet count.
To evaluate the relationships between liver volume and platelet counts, we followed the changes in LV/BW values according to the changes in platelet counts from baseline to 48 weeks after SVR; in the patients with increased and the patients with decreased platelet counts at 48 weeks after SVR (Figure 4). In patients with increased platelet counts, the LV/BW at SVR and 48 weeks later compared to baseline were 0.98 and 1.02, respectively. In patients with decreased counts at 48 weeks after SVR, the changes were 0.90 and 0.83, respectively. The changes at each point in LV/BW values of the patients with increased platelet counts were significantly greater than the changes in the values of the patients with decreased platelet counts (P = 0.017, P < 0.001, respectively). We followed the LV/BW value of 24 patients at 96 weeks after SVR. The rest of 12 patients were excluded due to lack of data or development of HCC (Figure 4). The LV/BW value in patients with decreased counts at 48 weeks increased from 0.83 to 0.91 at 96 weeks after SVR.

Discussion
In this study, we investigated the relationship between liver volume and the changes in platelet counts of Japanese patients with HCV infection treated with DAAs who obtained SVR. Following DAA therapy, not only spleen volume, but also liver volume was significantly decreased at SVR. Furthermore, the patients with decreased platelet counts at 48 weeks after SVR showed a greater reduction rate in liver volumes than patients with increased platelet counts. The reduction rate in liver volume was correlated with change in platelet count, while the reduction rate in spleen volume was correlated with change in albumin level. To the best of our knowledge, this is the first study to estimate changes in liver and spleen volumes by CT, and investigate the relationships between liver volume and liver function in Japanese patients with HCV treated by DAAs after achievement of SVR.
In this study, the spleen volume was continuously reduced until 48 weeks after SVR.
Spleen volume was reported to be related to the severity of fibrosis [16]. A previous study reported that platelet counts increased and spleen sizes decreased following IFN therapy [3]. Interventional management for splenomegaly by methods such as partial splenic artery embolization (PSE) or splenectomy leads to increased platelet counts and improved liver function [17]. In our study, the spleen volume decreased significantly, whether or not the platelet counts increased. However, change in spleen volume was significantly correlated with change in serum albumin level.
Studies have shown that reduction in spleen volume by PSE reduced portal pressure and flow in the splenic vein, but maintained portal perfusion [18][19][20]. These studies suggest that reduction in splenic arterial flow caused significant reduction in splenic venous flow with minimal decrease in the portal vein flow volume, while the flow of the superior mesenteric vein increased. The increased flow in the superior mesenteric vein led to improvement in liver function, including the level of serum albumin. We propose that the same mechanism underlies improvement in liver function after SVR achieved by DAA therapy.
Liver volume significantly decreased from baseline to SVR, and increased from 48 to 96 weeks after SVR in this study. The platelet counts correlated with the changes in liver volume and increased with liver volume. Chen et al reported that the volumes of the left, right, and caudate lobes tended to increase with exacerbation in inflammation and increased severity of fibrosis, but decreased in cirrhosis [11].
Another study reported that improvement in hepatic steatosis was accompanied by a significant decrease in the volumes of the left and right lobes, without significant change in spleen volume [21]. In our study, only 4 patients had fatty liver at baseline and the fatty liver of them were observed at 48 weeks after SVR too. For these reasons, the eradication of HCV by DAAs reduced hepatic inflammation, which might have led to reduction in liver volume.
Platelets play an important role in liver regeneration. A previous study reported that patients with low platelet counts after hepatic resection showed decreased regeneration of liver compared with patients with normal or high platelet counts [22]. Several studies described the following effects due to platelets that and cytokines such as IL-6, which are produced by macrophages in the spleen, might affect the progression of liver fibrosis and regeneration in patients with liver cirrhosis [17].
Decreased production of thrombopoietin (TPO) in the liver is another mechanism for thrombocytopenia. Cirrhotic patients with thrombocytopenia have lower levels of circulating TPO than patients without thrombocytopenia [27]. No published reports have mentioned an association between TPO and liver volume. However, since the liver is the main organ that produces TPO, a larger-volume liver might lead to higher levels of TPO.
In this study, the patients with decreased platelet count at 48 weeks after SVR showed greater reduction in liver volume than patients who increased that at SVR from baseline. The more inflammatory status, less reserved liver function or liver regenerating potential at baseline might affect the duration that re-increase the platelet counts and liver volume. Though it was not clarified in this study, portal hypertension may also affect these differences.
This study has limitations. This was a retrospective single-center study with insufficient numbers of participants in order to exclude the effect of antitumor therapy. Especially, the small sample size may over or underestimate the relationship between change in platelet count and change in LV/BW. The wide 95% confidential interval was also affected by small sample size. The measurement method of liver volume is also a limitation of this study. This study has risk of overestimate liver function because CT could not estimate how much of whole liver is function. We did not evaluate TPO or other liver regenerative factors at any time point. Additional studies that evaluate hematopoietic ability in relation to liver volume and platelet count are needed. Investigations are also needed to evaluate the association between liver volume and platelet count in patients with decompensated cirrhosis. And finally, further longitudinal studies are needed to investigate patient outcome including development HCC according to liver volumes or platelet counts.
In conclusion, the change in platelet count after SVR achieved by DAA therapy is

Consent for publication: Not applicable.
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: This study was not supported by any funding.
Authors' contributions: YS and YI contributed to the study design, data collection and analysis. YS was a major contributor in writing the manuscript. All authors contributed to data interpretation, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. All authors read and approved the final manuscript. Results are presented as n (%) for qualitative data or as median (range) for quantitative data.
Abbreviations: SVR, sustained virological response; BMI, Body mass index; HCC, hepatocellular carcinoma; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; AFP, alpha-fetoprotein.  The clinical parameters of patients treated by direct-acting antiviral (a. liver volume/body we Figure 3 Association between a. change in liver volume and platelet count, b. change in spleen volum The percent liver volume/body weight according to change in platelet counts from baseline t