Abstract
Background/Aim: The management of refractory ascites is critical for the treatment of patients with decompensated cirrhosis. This study aimed to evaluate the feasibility and safety of cell-free and concentrated ascites reinfusion therapy (CART) in patients with cirrhosis and refractory ascites, with a focus on changes in coagulation and fibrinolytic factors in ascitic fluid following CART. Patients and Methods: This was a retrospective cohort study including 23 patients with refractory ascites undergoing CART. Serum endotoxin activity (EA) before and after CART and the levels of coagulation and fibrinolytic factors and proinflammatory cytokines in original and processed ascitic fluid were measured. The Ascites Symptom Inventory-7 (ASI-7) scale was used for subjective symptom assessment before and after CART. Results: Body weight and waist circumference significantly decreased after CART, whereas serum EA did not significantly change after CART. Similar to the previous reports, ascitic fluid concentrations of total protein, albumin, high-density lipoprotein cholesterol, γ-globulin, and immunoglobulin G levels were significantly increased after CART; mild elevations in body temperature and interleukin 6 and tumor necrosis factor-alpha levels in ascitic fluid were also observed. Importantly, the levels of antithrombin-III, factor VII, and X, which are useful for patients with decompensated cirrhosis, were markedly increased in the reinfused fluid during CART. Finally, the total ASI-7 score was significantly lower following CART, compared with the pre-CART score. Conclusion: CART is an effective and safe approach for the treatment of refractory ascites that allows the intravenous reinfusion of coagulation and fibrinolytic factors in the filtered and concentrated ascites.
- Refractory ascites
- cell-free and concentrated ascites reinfusion therapy
- cirrhosis
- coagulation
- fibrinolytic
Cirrhosis, which is a terminal-stage liver disease, is associated with serious complications including refractory ascites resulting in decline in quality of life (1). Every year, approximately 10% of patients with cirrhosis develop refractory ascites despite treatment with fluid restriction, conventional diuretics, and the vasopressin-2 antagonist tolvaptan (2). The overall survival probability is very poor in patients with decompensated cirrhosis and refractory ascites, with an approximate rate of 30% at 2 years, and more than 50% of these patients develop hepatorenal syndrome (3, 4). Refectory ascites carry a high symptom burden with associated pain, nausea, need for hospital admission and limitation of function and mobility.
According to the Evidence-Based Clinical Practice Guidelines for Liver Cirrhosis 2020, resistance or poor response to diuretics is a clear indication for large-volume paracentesis (LVP) with albumin replacement or cell-free and concentrated ascites reinfusion therapy (CART) in patients with refractory ascites (5, 6). In simple paracentesis, hypoalbuminemia frequently develops as a high amount of protein is discarded (7). Potential pitfalls of LVP with albumin replacement include the inhibition of albumin synthesis and risk of infection (8), and appropriate use of albumin infusion prevents paracentesis-induced circulatory dysfunction (9). CART is difficult to conduct as ascites filtration and concentration is a complicated procedure. CART is associated with several adverse events, including shock, hypotension, chest pain, abdominal pain, dyspnea and hyperammonemia at puncture and drainage, and fever, chill, shivering, and hypotension (10). The Evidence-Based Clinical Practice Guidelines for Liver Cirrhosis 2020 suggests the infusion of 8 g/l albumin in therapeutic paracentesis in cases where the extracted ascites volume is ≥5 l. The guidelines also recommend paracentesis or CART for the treatment of refractory ascites (5, 6).
The National Medical Health Insurance does not fully cover the treatment cost for CART repeated sooner than 14 days in Japan. Nearly all studies investigating the efficacy and tolerability of CART are case series with no controls, and evidence is insufficient to establish the clinical efficacy of CART (11). CART is proposed to provide longer duration until re-drainage due to increased dietary intake and urine volume (12, 13). This study aimed to investigate the feasibility and efficacy of CART in patients with refractory ascites caused by decompensated cirrhosis.
Patients and Methods
The present study enrolled 23 patients with decompensated cirrhosis who received CART at the Department of Gastroenterology, Nara Medical University Hospital between June 1st 2020 and May 31th 2022. None of the following potential complications were present in the current study cohort: bleeding from the inferior epigastric artery, acute abdomen or abdominal wall hematoma, gastrointestinal perforation, entry site for pathogens, and peritonitis.
Laboratory data were examined several days prior to or on the day of CART and one day after the procedure. Activation of coagulation and fibrinolysis and levels of proinflammatory cytokines in the original and processed ascitic fluid samples were determined.
The Human Ethics Review Committee of Nara University Hospital approved the study protocol (Nara-Portal, 22-001), and the study was conducted according to the criteria set by the Declaration of Helsinki. All patients provided written informed consent to respond to a questionnaire survey and to provide blood samples before study enrollment.
CART. The standard CART includes three steps (14). First, ascites fluid is drained into a collection bag using abdominal paracentesis. In the present study, ascitic fluid was collected and processed at a rate of 50-100 ml/min and abdominal paracentesis achieved complete ascites drainage without complications. In the second step, bacteria, and cancer cells were removed via filtration and excess fluid and electrolytes were removed using the concentration method with an internal pressure-type system. In the final step, the filtered and processed ascites was reinfused (15). In the present study, a plasmapheresis device (KPS-8800Ce; Asahi Kasei Kuraray Medical, Tokyo, Japan) and circuit (KMT-8601; Kawasumi Laboratories, Tokyo, Japan) were utilized to process the ascitic fluid. Ascitic fluid evacuated using abdominal paracentesis was filtered through AHF-MO ascites filtration filters (Asahi Kasei Kuraray Medical), and the filtered ascitic fluid was concentrated using AHF-UP ascitic concentration filters (Asahi Kasei Kuraray Medical). Endotoxins were not measured in the collected ascitic fluid samples since CART cannot remove endotoxins (11). Total white blood cell (WBC) count in ascitic fluid was measured prior to CART, and absolute polymorphonuclear cell (PMN) count was calculated by multiplying the total WBC count by the percentage of polymorphonuclear cells. Patients with spontaneous bacterial peritonitis and an absolute PMN count of ≥250 cells/mm3 were excluded from the study. Infusion of processed ascitic fluid was initiated at a rate of 1.0 ml/min and maintained at a rate of 1.0-5.0 ml/min.
Evaluation of the coagulation and fibrinolytic cascades, EA, and proinflammatory cytokine levels. Whole-blood EA was measured using the commercially available Endotoxin Activity Assay kit (Spectral Diagnostics, Toronto, Canada), as previously described (16). Serum and ascitic fluid concentrations of tumor necrosis factor-alpha (TNFα) and interleukin-6 (IL-6) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Quantikine; R&D Systems, Minneapolis, MN, USA), according to the manufacturers’ instructions. Cytokine concentrations were calculated using standard curves. Commercially available ELISA kits were also used to determine serum and ascitic fluid concentrations of high-density lipoprotein cholesterol (HDL-C) (Wuhan Huamei Biotech, Wuhan, PR China) and immunoglobulin G (IgG) (Cat no. ab195215, Abcam PLC, Cambridge, UK). Antithrombin-III (AT-III) levels were measured using a chromogenic substrate method (17). Levels of factors VII and X and activity of proteins C and S were measured using clotting assays (18, 19).
The Ascites Symptom Inventory-7 scale. The ASI-7 scale was evaluated using a questionnaire administered before CART (Table I). The ASI-7 scale consists of seven items evaluated on a 5-point Likert scale as follows: 0, does not apply; 1, slightly applies; 2, somewhat applies; 3, strongly applies; and 4, very strongly applies. The questionnaire started with the following prompt: “Please answer the following questions about your ascites (abdominal fluid or fullness) and related symptoms, as they were on that day”. The seven items comprising the ASI-7 scale (Table I) included different domains and were arranged in a sequence from mild to severe symptoms representing clinical interpretation. The total ASI-7 scale score was determined by adding the score for each of the seven items, ranging from 0 to 4 points, for a total score ranging from 0 to 28 points. The ASI-7 scale scores exhibited an asymmetric distribution. The ASI-7 scale scores were used to categorize ascites as slight (0-11 points), mild (12-18 points), moderate (19-22 points), or severe (23-28 points).
Ascites symptom inventory-7 questionnairea.
Statistical analysis. Data were expressed as means±standard deviation. All statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA, USA) (20). Paired t-test was used for comparisons of two paired samples with normally distributed data. Wilcoxon’s signed-rank test was used for comparisons of two paired samples with non-normally distributed data. A two-tailed p-value of <0.05 was considered to indicate statistical significance.
Results
Baseline characteristics of patients with cirrhosis. Table II shows the baseline characteristics of 23 patients with cirrhosis included in the study. Briefly, the study cohort included 16 (69.6%) male patients with a mean age of 72.0±14.4 years. Within the cohort, 2 (8.7%), 4 (17.4%), 6 (26.1%), 7 (30.4%), 2 (8.7%), and 2 (8.7%) patients had Child-Pugh scores of 8, 9, 10, 11, 12, and 13, respectively; therefore, 6 (26.1%) and 17 (73.9%) patients had Child-Pugh class B and C cirrhosis, respectively. The causes of cirrhosis were hepatitis C virus, hepatitis B virus, alcohol intake and nonalcoholic steatohepatitis, in 6 (26.1%), 3 (13.0%), 9 (39.1%), and 5 (21.7%) patients, respectively. The rates of portal thrombosis and hepatocellular carcinoma were 13.0% (3/23) and 52.2% (12/23), respectively. The mean drained and processed ascitic fluid volumes were 5,700±1,964 and 295±168.6 ml, respectively. The mean reinfusion speed of processed ascites was 2.4±1.1 ml/min.
Baseline characteristics before concentrated ascites reinfusion therapy.
Changes in clinical indices after CART. Body weight and waist circumference were significantly decreased one day after CART (p<0.001 for both) (Table III). Although significant changes were observed in body temperature after reinfusion (p<0.001), the rise in body temperature was mild. Hemoglobin level, platelet count, and aspartate aminotransferase and alanine aminotransferase levels were significantly decreased one day after CART (p<0.001, p=0.0019, p=0.046, and p=0.031, respectively). No significant differences in systolic blood pressure or urine volume were observed after CART, indicating that CART had a minimal effect on hemodynamics. The levels of fibrin degradation products, fibrinogen, and D-dimer were significantly increased one day after CART (p<0.001, p=0.0018, and p=0.0019, respectively). However, no significant changes in WBC count and C-reactive protein levels were observed one day after CART (p=0.19, and p=0.099, respectively). Total protein (TP) and albumin levels remained unchanged after CART (p=0.74 and p=0.35, respectively).
Clinical indices before and 1 day after concentrated ascites reinfusion therapy.
Changes in serum and ascitic fluid cytokine levels and EA after CART. As previously reported, the TNFα and IL-6 levels were significantly higher in the processed ascitic fluid compared to the original ascitic fluid (p<0.001 and p=0.009) (Figure 1A and B). However, no significant differences in serum TNFα and IL-6 levels were found between the pre- and post-CART samples (p=0.40 and p=0.96, respectively) (Figure 1C and D). No significant difference in EA levels was found between the pre-CART whole-blood samples and those collected immediately after CART (p=0.89) (Figure 2).
Ascitic fluid concentrations of (A) tumor necrosis factor-alpha (TNFα) and (B) interleukin-6 (IL-6) before and after cell-free and concentrated ascites reinfusion therapy (CART). Serum concentrations of (C) TNFα and (D) IL—6 before and after CART.
Serum endotoxin activity before and after cell-free and concentrated ascites reinfusion therapy (CART).
Changes in laboratory parameters after CART. The concentrations of TP, albumin, γ-globulin, HDL-C, IgG, AT-III, factors VII and X, and proteins C and S were markedly increased in the processed ascitic fluid compared to the original ascitic fluid (p<0.001 for all) (Figure 3). No significant changes in serum concentrations of these parameters were observed between the pre- and post-CART ascitic fluid samples (data not shown).
Ascitic fluid concentrations of specific proteins and coagulation and fibrinolytic factors before and after cell-free and concentrated ascites reinfusion therapy (CART). A) Total protein, B) albumin, C) γ-globulin, D) high density lipoprotein-cholesterol (HDL-C), E) immunoglobulin G (IgG), F) antithrombin-III, G) factor VII, H) factor X, I) protein C, and J) protein S.
Changes in ASI-7 scores after CART. The total ASI-7 score and the scores for items 1, 2, 3, 4, 6, and 7 were significantly decreased after CART (Figure 4 and Figure 5).
Total Ascites Symptom Inventory-7 (ASI-7) scores before and after cell-free and concentrated ascites reinfusion therapy (CART).
Scores for each of the seven items in the Ascites Symptom Inventory 7 (ASI-7) scale before and after cell-free and concentrated ascites reinfusion therapy (CART).
Comparison of specific protein levels between the original and processed ascites. Table IV shows the levels of specific proteins in the original and processed ascitic fluid samples. The recovery rates of specific proteins in the ascitic fluid were examined. The recovery rates of TP, ALB, γ-globulin, HDL-C, and IgG were 80.0±14.7%, 80.0%±11.7%, 82.0%±11.2%, 75.9%±21.2%, and 75.0%±20.1%, respectively.
The levels of total protein and albumin in original and processed ascites.
Discussion
CART is a clinically effective and safe approach to improve subjective symptoms and reduce the risk of infection of patients with cirrhosis. However, few studies have evaluated the coagulation and fibrinolytic systems in patients with cirrhosis undergoing CART (21, 22). In the present study, we found that CART led to marked increases in the levels of activation products of the coagulation and fibrinolytic cascades, such as AT-III, in patients with cirrhosis.
In the present study, all patients developed fever after the reinfusion of the processed ascitic fluid. However, no significant change in EA was observed between the pre- and post-CART whole-blood samples. Elevated body temperature can be triggered by febrile non-hemolytic transfusion reactions, which occurs in 1%-3% of transfusions (23). Fibrin has been shown to cause fever in cases where processed ascitic fluid is administered by intravenous infusion (24). We recently reported proinflammatory cytokines IL-6 and TNF-α in ascitic fluid as pyrogenic substances during CART (14). In contrast, the IL-6 concentrations were reported to decrease with filtration and concentration processes during CART (25). While the reason for the discrepancy in proinflammatory cytokine levels remains unclear, differences in the reinfusion rate of the processed ascitic fluid across the studies are a possibility (10) (26, 27). The infusion of IL-6 over a short period of time stimulates immune responses such as a rise in body temperature (25). Therefore, the potential pyrogenic role of cytokines and fibrin in CART warrants further investigation.
Serum TP and albumin levels were significantly elevated after CART compared with the pre-CART values (26). Consistent with our findings, several studies reported that the serum levels of TP and albumin levels after CART were comparable to those before CART (11, 28). In one study, no significant changes in serum TP and albumin levels were observed in 11 patients with decompensated cirrhosis who underwent CART for a total of 24 times (11). Conversely, CART leads to significant increases in serum albumin levels compared to paracentesis (12). In a recent study, Hanai et al. showed that CART, LVP, and CART in combination with LVP exhibited comparable therapeutic effect in patients with refractory ascites (29). Although the cause underlying the differences in post-CART serum albumin levels among the studies remains unclear, it is possible that the total albumin levels in the processed ascitic fluid were different among the studies. These findings indicate that CART exerts beneficial effects on the maintenance of serum albumin levels because serum albumin level gradually decreases over time in patients with decompensated cirrhosis. In this study, the average albumin level in processed ascites was 21.7 g, which is considerably lower than 75 g, which is the cutoff value that has to be reached to obtain full coverage of CART by medical insurance in Japan.
Clinical practice guidelines issued by the American Association for the Study of Liver Diseases (30) and the European Association for the Study of the Liver (31) recommend the infusion of human albumin after paracentesis and emphasize the need for albumin substitution (6-8 g per liter of fluid removed) in patients with a ≥5 l drained volume (32). In Japan, 8 g/L albumin is recommended for substitution in therapeutic paracentesis with a drained ascites volume of ≥5 l; therefore, CART provides an advantage of reinfusing coagulation and fibrinolytic factors including AT-III (5, 6). In addition, the pathophysiology of portal vein thrombosis (PVT) in cirrhosis consists of decreased portal flow and hypercoagulable state (33). In Japan, AT-III supplementation therapy has been used in patients with PVT and a serum AT-III level of ≤70% (34). During CART, coagulation and fibrinolytic factors are activated in ascitic fluid and may re-enter the systemic circulation and induce systemic anticoagulation and hyperfibrinolysis (35).
Nevertheless, we acknowledge the limitations of our study. First, the sample size was very small. Second, the study did not include a control group of patients who only underwent abdominal paracentesis. Therefore, randomized controlled trials are warranted to obtain higher level evidence on the reinfusion of coagulation and fibrinolytic factors in patients with cirrhosis undergoing CART.
Conclusion
In conclusion, the present study findings suggest CART as a safe and feasible treatment for patients with refractory ascites. The ultimate goal of CART is to relieve tense ascites and to reinfuse albumin as well as coagulation and fibrinolytic factors to prevent further complications, such as PVT, which are associated with high mortality.
Acknowledgements
The Authors would like to thank Enago for English editing and proofreading of the article.
Footnotes
Authors’ Contributions
Conceptualization, H.Y.; Methodology, T.A., A.M, Y.Fujina, M.Y. and K.Ka.; Validation, K.Mu.; Investigation, T.K., S.I., F.T., and S.T.; Resources, J.S, M.E, H.Ta., H.K., and Y.T; Data curation, Y.Fujimo., N.N, A.S., and K.Ki.; Formal analysis, T.I.; Writing—original draft preparation, N.Y.; Writing—review and editing, T.N and H.Y. All Authors have read and approved the manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received February 4, 2023.
- Revision received February 16, 2023.
- Accepted February 17, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
