Splenic artery steal syndrome in patients with orthotopic liver transplant: Where to embolize the splenic artery?

PURPOSE: This study compared proximal and distal embolization of the splenic artery (SA) in patients with splenic artery steal syndrome (SAS) after orthotopic liver transplantation (OLT) regarding post interventional changes of liver function to identify an ideal location of embolization. METHODS AND MATERIALS: 85 patients with SAS after OLT treated with embolization of the SA between 2007 and 2017 were retrospectively reviewed. Periinterventional DSA was used to assess treatment success and to stratify patients according to the site of embolization. Liver function was assessed using following laboratory values: bilirubin, albumin, gamma-glutamyl transferase, glutamat-pyruvat-transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), Alkaline Phosphatase (ALP), aPTT, prothrombin time and thrombocyte count. Descriptive statistics were used to summarize the data. Median laboratory values of pre, 1- and 3-days, as well as 1-week and 1-month post-embolization were compared between the respective embolization sites using linear mixed model regression analysis.
RESULTS: All procedures were technically successful and showed an improved blood flow in the hepatic artery post-embolization. Ten Patients were excluded due to re -intervention or inconsistent image documentation. Pairwise comparison using linear mixed model regression analysis showed a significant difference between proximal and distal embolization for GPT (57.0 (IQR 107.5) vs. 118.0 (IQR 254.0) U/l, p = 0.002) and GOT (48.0 (IQR 48.0) vs. 81.0 (IQR 115.0) U/l, p = 0.008) 3-days after embolization as well as median thrombocyte counts 7-days after embolization (122 (IQR 108) vs. 83 (IQR 74) in thousands, p = 0.014). For all other laboratory values, no statistically significant difference could be shown with respect to the embolization site.
CONCLUSION: We conclude that long-term outcomes after embolization of the SA in the scenario of SAS after OLT are irrespective of the site of embolization of the SA, whereas a proximal embolization potentially facilitates earlier normalization of liver function. Choice of technique should therefore be informed by anatomical conditions, safety considerations and preferences of the interventionalist.

Introduction

For patients suffering from complex liver diseases such as advanced stages of cirrhosis, certain stages of liver cancer and congenital or acquired abnormalities in anatomy or metabolism of the liver, orthotopic liver transplantation (OLT) is usually the only curative treatment option. In this context, splenic artery steal syndrome (SAS) is a frequently described vascular complication after OLT with incidences reported between 3 and 6% in the literature [1-5]. SAS describes a pathological redirection of blood flow from the hepatic artery towards the splenic artery (SA) resulting in reduced hepatic arterial perfusion in the absence of an occlusion of the hepatic artery. This steal-situation might lead to ischemia, graft dysfunction and biliary damage, resulting in organ failure [2, 6, 7]. The clinical picture is non-specific which makes the diagnosis of SAS challenging [2, 8]. If clinically suspected, patients usually undergo a digital subtraction angiography (DSA) scan of the celiac axis. SAS is diagnosed by confirmation of an enlargement of the SA in combination with late enhancement of the hepatic artery territory [2, 9].

In this regard, embolization of the SA has been reported to be a safe and effective treatment to redirect blood flow to the hepatic circulation [2, 7, 10–12]. Better perfusion of the transplant graft after embolization is mainly due to two main effects. Firstly, it results in redirection of blood flow from the splenic flow territory to the hepatic artery territory increasing blood pressure in the common hepatic artery. Secondly, another mechanism has been discussed in the literature called hepatic artery buffer response (HABR) [13, 14]. It describes a reciprocal relationship between blood flow in the hepatic artery and the portal vein. A decrease of flow to the spleen automatically results in a lower blood volume in the portal vein. Through a negative feedback mechanism this finally leads to vasodilatation of hepatic arteries and an improved arterial blood flow to the liver. Embolization is commonly done using coils [10] or Amplatzer venous plugs [11], inserted into the SA via an intraarterial catheter until complete stasis is seen on DSA. However, there are risks and challenges associated with different approaches for embolotherapy whereas the ideal strategy is commonly informed by the target site. Data on the impact of the embolization site is scarce and a potentially ideal location in regard to graft function has not yet been evaluated [4, 11, 15, 16].

This study retrospectively investigated the possible superiority of either proximal or distal embolization of the SA in patients suffering from SAS after OLT regarding post-interventional changes of liver function.

Methods and materials

Patient selection

This retrospective cohort study was conducted in the radiological department of *blinded*. All patients provided written informed consent and the study was approved by the local ethics committee. Between November 2007 and January 2017, a total of 85 patients with SAS after OLT were referred to our department of interventional radiology for embolization of the SA. Inclusion criteria for this study were: (1) Embolization of the SA due to SAS, (2) Follow-up at least for 30 days after embolization and (3) no rejection of the transplant or major vascular complications during follow-up. A total of ten Patients had to be excluded after the initial search due to following reasons: Five patients had missing or incomplete image documentation which made it impossible to determine the exact location of embolization. Two patients had to undergo re-OLT due to transplant rejection within the first 30 days of OLT. Completely missing laboratory values lead to exclusion of two more patients. One patient received stenting of the hepatic artery in the same procedure in which embolization of the SA was performed because of a suspected partial dissection of the vessel. This patient was also excluded, leaving a total of 75 patients who were included into the final data analysis. Fig 1 shows the process of patient selection and -exclusion in a flowchart.

Fig 1

Exclusion flowchart.

A total of ten patients were excluded. 75 patients were included in the final data analysis. Of these 75 patients a total of 60 patients had complete laboratory data during the 30-days follow up. 15 patients missing data but could be included into the linear mixed model regression analysis. SAS, Splenic Artery Steal Syndrome; SA, Splenic Artery; OLT, Orthotopic Liver Transplantation.

Diagnosis of SAS

All diagnostics were based on the current understanding within the scientific community. Since SAS is a diagnosis of exclusion, patients were diagnosed in a stepwise approach [2, 7, 8, 17]. Initially, SAS was suspected due to elevated laboratory levels of transaminases, Alkaline Phosphatase (ALK) and Bilirubin or persistent ascites in the absence of acute cellular rejection, infection or toxicity.

Patients were then further examined with Doppler ultrasound. Dynamic assessment of velocity, waveforms, and resistance index (RI) of the abdominal arteries helps differentiate SAS from other causes of graft hypoperfusion such as hepatic artery stenosis or hepatic artery thrombosis. Patients suffering from SAS usually show high RIs (>0.8) in the hepatic artery with low diastolic flow or even reversal of diastolic flow. In addition, hepatic artery systolic velocities are unusually low (<35 cm/s) typically showing weak blood flow in the hepatic artery [17]. The diagnosis was confirmed if both, relative arterial hypoperfusion of the graft and an enlarged splenic artery was seen on DSA. Typical dynamic angiographic findings are (1) early perfusion of the splenic or gastroduodenal artery together with (2) delayed or dim perfusion of the hepatic artery along with (3) early portal venous contrast filling indicating hampered hepatic arterial blood flow (Fig 2A–2C) [7].

Fig 2

Abdominal DSA.

A-C. Pre-intervention celiac angiography shows hampered hepatic arterial flow with delayed contrast filling of the hepatic artery (blue arrows) as well as early and strong contrast filling of the enlarged splenic artery. B-D. Post-interventional celiac angiography shows significantly improved hepatic arterial flow and delayed contrast filling of the splenic artery. B. shows embolization of the SA using an Amplatzer venous plug (red marker), D. shows an embolization using coils (green marker).

Procedure techniques

Board-certified radiologists all with > 10 years of experience, performed the interventions. After local anesthesia with lidocaine 1%, vascular access in micropuncture technique was performed preferably in the right common femoral artery or left brachial artery and a 6F vascular sheath was placed in Seldinger-technique via a 0.035-inch guide wire. An overview angiography of the celiac trunk and the superior mesentery artery was generated with a 5-F Cobra (Radifocus, Terumo Europe NV) or a 5-F SOS Omni Selective catheter (Soft-Vu, Angiodynamics) before advancement into the splenic artery (Fig 2A–2C). Additional DSA runs allowed for analysis of splenic artery anatomy and size. The catheter was advanced into a favorable position. If advancement of the macrocatheter was not possible, the SA was accessed via a microcatheter (Cantata 2.5 F or MicroFerret-18 3 F, Cook Medical, Bloomington, IN). For embolization with either an Amplatzer plug (AGA Medical, Golden Valley, MN) a 6F curved sheath was introduced into the SA (Fig 2B). Coil embolization was either done using the macro- or microcatheter (Fig 2D). Gianturco coils of variable of various sizes (Cook Medical, Bloomington, IN) were used. Embolization was performed until stasis of blood flow was seen on DSA (Fig 2B–2D). Following OLT, all patients received a calcineurin-inhibitor-based immunosuppressive protocol and steroids which are usually tapered within the first month after surgery. NSAR or antibiotics are not part of our standard of care for SA embolization but may have been administered at discretion of the referring department depending on the clinical scenario.

Periinterventional DSA was retrospectively used to assess treatment success and to stratify patients according to the site of embolization. Liver function was assessed using following laboratory values: bilirubin (TBIL), albumin, gamma-glutamyl transferase (GGT), glutamat-pyruvat-transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), ALP, aPTT, prothrombin time (PT) using international normalized ratio (INR) and thrombocyte count.

Statistical analysis

Descriptive statistics were used to summarize the data in absolute numbers and percent. Testing for normality was performed with the Shapiro Wilk test. Non-normally distributed data are expressed as median and interquartile range (IQR) and compared with the nonparametric Mann-Whitney U test and Wilcoxon signed-rank test. Median laboratory values of pre, one-, 3-days, as well as 1-week and 1-month post embolization were compared. Linear mixed model regression analysis was used to identify significant effects of the embolization site on repeated measurements of the above-mentioned laboratory values across different time points. Conception of the longitudinal mixed model was informed by the impact on the Akaike’s and Bayesian information criteria which both penalize for the complexity of a given model in order to minimize overfitting when explaining the variance in the data. For the model used in this study, time was treated as a categorial variable. The models included the embolization site, time, baseline scores, age as well as embolization site * time interactions as fixed covariates. The intercept was modeled as random effect. We expected the correlation between measurements to decrease with increased time between the measurements which is why an autoregressive co-variance structure was chosen. The results were normalized through transformation with the natural logarithm to allow for pairwise comparison with the standardized mean difference (SMD) and visualization with Forest plots. All statistical analyses were performed using IBM SPSS STATISTICS, version 25 (IBM Corporation, Armonk, NY, USA). Hypothesis tests were 2-tailed and used a 5% significance level.

Results

Median patient age of the patient cohort was 52.8 (IQR 13) years. Complete documentation of laboratory values after 30 days was available for 60 patients, incomplete data was available for a total of 75 patients. After 30 days, all included patients were alive. Table 1 summarizes patient characteristics of our cohort. In most patients (81%, n = 61), a femoral access was used, a brachial access was used in only 19% (n = 14) of cases. Embolization was done using Amplatzer venous plugs in 76% (n = 57) and coils in 11% (n = 7) of cases. In 13% (n = 11) a combination of both was used. A proximal site of embolization of the SA was chosen in 55% (n = 41) of cases. All procedures were technically successful resulting in an improved blood flow in the common hepatic artery post-embolization by means of angiographic criteria [7]. Of note, none of the patients suffered from a procedure related complication such as infections or relevant peri-interventional pain.

Table 1

Patient characteristics.

Parameter		N (%)
Demographics
Number of patients		75 (100)
Age	Median (IQR)	52.8 (13)
	>60 years	25 (30)
	<60 years	50 (70)
Sex (female) (%)		27/75 (36%)
Indication for OLT	Alcohol liver cirrhosis	28 (37)
	Cryptogene cirrhosis	20 (27)
	HCC	17 (23)
	HCV cirrhosis	9 (12)
	Primary biliary cirrhosis	1 (1)
Treatment
Site of embolization	proximal	41 (55)
	distal	34 (45)
Access	femoral	61 (81)
	radial	14 (19)
Embolization Material	Coils	7 (11)
	Amplatzer Plaque	57 (76)
	Coils + Plaques	11 (13)
Median time from OLT to treatment in days (IQR)		44 (53.5)

SD, standard deviation; OLT, orthotopic liver transplantation; HCC, hepatocellular carcinoma; HCV, hepatitis C virus.

Using the Wilcoxon signed-rank-test for the comparison of dependent measurements at the beginning and the end of the observational period for 63 patients with complete data, a significant decrease of median laboratory values over the course of 30 days could be detected for TBIL by 50.2% (2.95 (IQR 6.94) to 1.47 (IQR 3.06) mg/l), p = 0.003, 78% of patients showing normalized values), GPT by 47.5% (80 (IQR 208) to 42 (IQR 39) U/l), p<0.001, 83% of patients showing normalized values), GOT by 47.7% (66 (IQR 365.2) to 34.5 (IQR 48.5) U/l), p<0.001, 42% of patients showing normalized values), and PT by 4.4% (1.26 (IQR 0.33) to 1.21 (IQR 0.28), p = 0.013, 72% of patients showing normalized values). A significant increase could be shown for albumin by 4.3% (30.1 (IQR 6.2) to 31.3 (IQR 7.7) g/l, p = 0.002, 61% showing normalized values), for ALP by 66.1% (121 (IQR 121) to 201 (IQR 231) U/l), p = 0.001, 47% showing normalized values) and for thrombocytes count by 62.5% (80T (IQR 127) to 130T (IQR 100.5), p<0.001, 66% showing normalized values after 30 days), respectively. Changes of laboratory values were non-significant after 30 days for GGT (+9.1% (175 (IQR 225) to 159 (IQR 229) U/l), p = 0.341) and aPTT (-10.7% (36.8s (IQR 8.2) to 40.7s (IQR 13.6), p = 0.07, 33% of patients showing normalized values). Fig 3 shows the development of median laboratory values over time stratified regarding the site of embolization.

Fig 3

Boxplots showing the development of median laboratory values over time stratified regarding the site of embolization.

GGT, gamma-glutamyl transferase, GPT, glutamic-pyruvic transaminase; GOT, glutamic-oxaloacetic transaminase; ALP, Alkaline Phosphatase; PT, Prothrombin Time using International Normalized Ratio (INR).

According to the Mann-Whitney U test, there were no statistically significant differences of median laboratory values between the cohorts of proximal and distal location of embolization at any time point. Notably, no significant difference could be detected between both patient groups at baseline. The results of the longitudinal mixed model analyses along with forest plots are reported in Fig 4. Pairwise comparison revealed a significant difference between proximal and distal embolization for GPT (57.0 (IQR 107.5) vs. 118.0 (IQR 254.0) U/l, p = 0.002) and GOT (48.0 (IQR 48.0) vs. 81.0 (IQR 115.0) U/l, p = 0.008) 3-days after embolization. Furthermore, a significant difference could be detected for median thrombocyte counts 7-days after embolization (122 (IQR 108) vs. 83 (IQR 74) in thousands, p = 0.014). For all other laboratory values, no statistically significant difference could be shown between both groups stratified regarding the site of embolization.

Fig 4

Median values as well as pairwise comparisons differentiated after location of embolization of the SA.

* The mean difference is significant at the .05 level. IQR, interquartile range; SA, splenic artery; CI, confidence interval; GGT, gamma-glutamyl transferase, GPT, glutamic-pyruvic transaminase; GOT, glutamic-oxaloacetic transaminase; ALP, Alkaline Phosphatase; PT, Prothrombin Time. Forest plot analysis of the log-transformed standardized mean difference (SMD) for pairwise comparison.

Discussion

This study could show that a successful embolization of SAS after OLT is independent of the localization of embolization of the SA.

In detail our results draw two main findings: Firstly, as evaluated by laboratory values, embolization of the SA is an effective treatment of SAS to improve function of the OLT. Secondly, although we could encounter statistically significant differences between proximal and distal embolization for GPT, GOT and for thrombocyte counts at single time points, for the vast majority of laboratory values and time points no statistically significant difference could be shown. Moreover, laboratory values tend to match after 30 days indicating a self-adjusting equilibrium.

Coil and Amplatzer venous plug embolizations are well described in the literature and have been successfully implemented in daily clinical routine [4, 7, 11, 18]. However, several limitations have been reported for positioning of embolic material with multiple cases of dislocation and splenic infarction as a result of far distal embolization [4, 16, 19].

Besides operator preference for embolic materials the question of where to embolize is not easy to answer. As stated in the literature, more proximal occlusion of the SA lowers the risk of infarction of the spleen by perfusion through arteries from the gastric, pancreatic, and gastroepiploic territory, hence preserving a sufficient perfusion of the spleen [15]. Our data shows, that a more proximal embolization is associated with significantly higher thrombocyte counts 3-days post intervention. This might be due to an overall better perfusion of the spleen and hence, a mostly preserved platelet physiology [20]. The statistically significant, transient increase in GPT and GOT and the not significant increase in ALP and bilirubin levels after distal embolization appear to be in certain contrast to the more consistent down-trending of the aforementioned parameters after proximal embolization. This might indicate a transient or rather prolonged liver hypoperfusion after distal embolization but a similar long-term outcome, presumably due to autoregulation.

Especially in situations of daily clinical routine an embolization of proximal parts of the SA might be more feasible and faster without the use on microcatheters [11] and allows for the use of both, Amplatzer venous plugs and coils as used in parts of our study cohort [7, 11, 16]. Our results encourage interventionalists to be free to use whatever material they are most familiar with. We believe this might ultimately result in higher success rates and better patient outcomes. To our knowledge, this is the first study that assessed the impact of the embolization site with respect to changes of liver-related laboratory values over time.

This study has several limitations. It is a single-institutional retrospective analysis of 75 patients with SAS after OLT treated with embolization of the SA. The medium-sized cohort with fairly heterogeneous underlying diseases lacks subgroup analysis and might therefore not be representative. Being one of the largest transplant centers in Germany however, our study includes all patients that were treated in our institution and it will be challenging to find a larger cohort without endangering the consistency of the study protocol. Due to inconsistent follow-up imaging a volumetric assessment of the size of the spleen was not possible. At this point we’re conducting further studies to evaluate changes of spleen volume after embolization in order to predict successful treatment of SAS.

Conclusion

This study could show that long-term outcomes after embolization of the SA in the scenario of SAS after OLT are independent of the localization of embolization of the SA, whereas a proximal embolization potentially facilitates earlier normalization of liver function. Choice of embolization technique and site might therefore solely be based on interventionalists preferences and anatomical conditions.

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20 Sep 2021

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Reviewer #1: The authors presented a retrospective evaluation of the splenic artery embolization in patients with steal syndrome after orthotopic liver transplantation. They compared the effect of proximal and distal embolization and they concluded that the site of the embolization had no impact on the beneficial effect of the intervention. The conclusion is based only on the comparison of the course of laboratory parameters during the 30 days after the procedure. The paper might bring new interesting facts but the data should be better described and interpreted. The manuscript needs a major revision to be suitable for publication.

Major comments

1. The patient's selection process must be better explained. There were 85 patients who underwent SA embolization for SASS but 10 of them were excluded from further evaluation because of the need of re-intervention which was not related to the SA embolization. However, the term re-intervention implicates the sensation that the patients needed the same procedure again. This point should be explained in detail. Were there patients with inefficient first embolization procedure? If yes, how were they distributed between the proximal and distal embolization site subgroups?

2. The laboratory data during the observation period were available only in 60 patients. The numbers of the evaluated patients must be added in the appropriate columns in the table 2. A flow chart showing the number of included, excluded and evaluated patients might be helpful for better understanding the results.

3. The process leading to the SAS diagnosis is unclear. The authors wrote that patients had had an alteration in specific liver laboratory values. When looking at figure 2, it seems that there were also patients with normal laboratory values at day 0. What was the role of ultrasound in the decision to perform DSA? Were there also patients with weak hepatic artery blood flow on ultrasound but with normal laboratory parameters?

4. The authors should better document the improvement of liver graft function after the SA embolization in each subgroup of patients. They should add the proportions of patients who normalised GPT and other enzymes or achieved normal platelet count after the 30-days observation period.

Minor comments

Some abbreviations were used incorrectly in the manuscript:

1. The authors defined only abbreviation SA for splenic artery, but they used also SAE for splenic artery embolization. SAE represents a generally accepted abbreviation for serious adverse event. I would recommend not to use this abbreviation for a different term.

2. GOT and AST are two abbreviations for the same enzyme (Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or glutamic oxaloacetic transaminase (GOT, SGOT), is a pyridoxal phosphate (PLP)-dependent transaminase enzyme (EC 2.6.1.1) but they were presented in the text and also in the table 2 and figure 2 as two different enzymes.

3. AP represents the abbreviation for the enzyme alkaline phosphatase (EC 3.1.3.1). The values of AP were presented in the section Results, but it was not mentioned in the section Procedure techniques. Furthermore, the abbreviation AP is missing in the comment under the table 2 and figure 2.

4. INR (international normalized ratio) is a standardised method for the expression of prothrombin time results. The assessed parameter should be called prothrombin time (PT) and INR as the mode of measurement (unit).

5. The authors used SAS and also SASS without explaining the difference between both abbreviations.

Reviewer #2: In a retrospective cohort study, the authors compared proximal versus distal splenic artery embolization in patients with splenic artery steal syndrome after liver transplantation with respect to postinterventional changes in surrogate markers of liver function. This study is of importance because the optimal site for embolization is unclear and few data are available on this topic. They observed a median increase in GPT levels after distal embolization but a decrease after proximal embolization and improvement in platelet counts at 3 and 7 days. The manuscript is well written and the data are well presented.

Key Points:

Methods: Please explain in more detail the use of the mixed linear regression model (fixed and random effects). Why do the authors believe this model is superior to the nonparametric comparison of laboratory values at time 0 and time x using the signed-rank test?

Figure 2: To better assess the change in transaminase levels after proximal versus distal embolization, I propose to plot median delta transaminase levels (compared with day 0) for all time points.

Do the authors have data on white blood cell counts or CRP levels after embolization to assess whether systemic inflammation differs between proximal and distal embolization?

The increase in GPT levels at 3 days alongside similar levels at 30 days could indicate transient liver hypoperfusion after the procedure but a similar long-term outcome. Please discuss.

Minor points:

The authors state that there were no procedure-related complications. Does this apply to pain, splenic abscesses, and infections?

Were patients routinely treated with nonsteroidal anti-inflammatory drugs, steroids, or antibiotic prophylaxis?

Please provide any other characteristics of the study cohort, including time since transplant and immunosuppression, that might affect transaminase progression.

Was hepatic artery stenosis excluded in all patients?

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3 Jan 2022

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors presented a retrospective evaluation of the splenic artery embolization in patients with steal syndrome after orthotopic liver transplantation. They compared the effect of proximal and distal embolization and they concluded that the site of the embolization had no impact on the beneficial effect of the intervention. The conclusion is based only on the comparison of the course of laboratory parameters during the 30 days after the procedure. The paper might bring new interesting facts but the data should be better described and interpreted. The manuscript needs a major revision to be suitable for publication.

Major comments

1. The patient's selection process must be better explained. There were 85 patients who underwent SA embolization for SASS but 10 of them were excluded from further evaluation because of the need of re-intervention which was not related to the SA embolization. However, the term re-intervention implicates the sensation that the patients needed the same procedure again. This point should be explained in detail. Were there patients with inefficient first embolization procedure? If yes, how were they distributed between the proximal and distal embolization site subgroups?

We fully agree with the reviewer and added a paragraph regarding the exclusion of patients during the selection process. Five patients were excluded due to missing/incomplete imaging, two because of missing lab values as well as re-OLT after organ rejection. One patient received a stent placement in the hepatic artery synchronous with the embolization procedure. Indication for this was a partial dissection of the hepatic artery. Please see also figure 1 where we clarified the selection process in a flow chart diagram as suggested in comment 2.

2. The laboratory data during the observation period were available only in 60 patients. The numbers of the evaluated patients must be added in the appropriate columns in the table 2. A flow chart showing the number of included, excluded and evaluated patients might be helpful for better understanding the results.

Thank you very much for this comment. 60 patients had all laboratory data available at all time points. Out of 75 patients a total of 15 patients had at least one laboratory value missing. Yet, linear mixed model regression analysis allowed us to include patients with missing values into the analysis to strengthen our data. In fact, this was the reason why we chose this statistical model over a simpler repeated measures ANOVA approach. Hence, we were able to include all 75 patients into the statistical analysis. We clarified this in the methods and results section in order to explain this to potential readers. Also thank you very much for the suggestion of a flow chart. Please see figure 1. We believe this figure clarifies also parts of comment 1.

3. The process leading to the SAS diagnosis is unclear. The authors wrote that patients had had an alteration in specific liver laboratory values. When looking at figure 2, it seems that there were also patients with normal laboratory values at day 0. What was the role of ultrasound in the decision to perform DSA? Were there also patients with weak hepatic artery blood flow on ultrasound but with normal laboratory parameters?

This is a very helpful comment by the reviewer,. SAS is a diagnosis of exclusion and although conventional angiography is necessary in the diagnosis of SAS, no true gold standard for imaging exists. The diagnosis is suspected based upon a constellation of clinical, laboratory, and imaging findings after exclusion of more common causes of graft dysfunction. Ultimately, the diagnosis of SAS is made ex juvantibus when increased hepatic arterial perfusion and improved graft function are seen after splenic artery embolization.

Hence, we use a stepwise approach to narrow down the differential diagnosis before any intervention. After exclusion of cellular rejection, infection or toxicity, Doppler ultrasound is used to rule out vascular and biliary complications in OLT patients. Evaluation of hepatic artery velocity, waveforms, and particularly vascular resistance helps differentiate SAS from Hepatic Artery Thrombosis and Hepatic Artery Stenosis with collateralization. However, most findings are non-specific and could be caused by transient graft edema, organ rejection, or infection.

Like assumed by the reviewer, there were indeed patients showing normal laboratory values at baseline. Yet, even if some laboratory values were normal, they were always accompanied by abnormalities of other surrogate parameters of liver function. Besides, in some patients Doppler Ultrasound was also performed if persistent ascites was seen in patients without any other cause for this such as rejection or infection. We clarified our diagnostic workflow in the methods part of the manuscript with emphasis on the stepwise approach including the role of Doppler Ultrasound.

4. The authors should better document the improvement of liver graft function after the SA embolization in each subgroup of patients. They should add the proportions of patients who normalized GPT and other enzymes or achieved normal platelet count after the 30-days observation period.

Thank you for this comment. We added the percentage of normalized laboratory values after 30 days in the results section for every value evaluated. Although all procedures were technically successful, we were not able to achieve an absolute normalization in all values. Yet, when looking at the statistical analysis we could achieve a significant improvement of all laboratory values but GGT in the 30-days post-intervention. We clarified this in the results section of the manuscript.

Minor comments

Some abbreviations were used incorrectly in the manuscript:

1. The authors defined only abbreviation SA for splenic artery, but they used also SAE for splenic artery embolization. SAE represents a generally accepted abbreviation for serious adverse event. I would recommend not to use this abbreviation for a different term.

We standardized our abbreviations accordingly throughout the manuscript. For example, “embolization of the SA” is simply used instead of SAE.

2. GOT and AST are two abbreviations for the same enzyme (Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or glutamic oxaloacetic transaminase (GOT, SGOT), is a pyridoxal phosphate (PLP)-dependent transaminase enzyme (EC 2.6.1.1) but they were presented in the text and also in the table 2 and figure 2 as two different enzymes.

Thank you for this comment. We corrected double use of the two names for the same enzyme in all sections of the manuscript including abbreviation section of figures and tables.

3. AP represents the abbreviation for the enzyme alkaline phosphatase (EC 3.1.3.1). The values of AP were presented in the section Results, but it was not mentioned in the section Procedure techniques. Furthermore, the abbreviation AP is missing in the comment under the table 2 and figure 2.

We added alkaline phosphatase in the methods and material part of the manuscript as well as in table 2 and figure 3. We furthermore changed the abbreviation AP to ALP which is the more commonly used term in English literature. Thank you very much!

4. INR (international normalized ratio) is a standardised method for the expression of prothrombin time results. The assessed parameter should be called prothrombin time (PT) and INR as the mode of measurement (unit).

We implemented prothrombin time for the assessed parameter in the manuscript as well as in all tables and figures

5. The authors used SAS and also SASS without explaining the difference between both abbreviations.

There is no difference, we corrected the abbreviation and standardized using the term SAS for “splenic artery steal syndrome”

Reviewer #2: In a retrospective cohort study, the authors compared proximal versus distal splenic artery embolization in patients with splenic artery steal syndrome after liver transplantation with respect to postinterventional changes in surrogate markers of liver function. This study is of importance because the optimal site for embolization is unclear and few data are available on this topic. They observed a median increase in GPT levels after distal embolization but a decrease after proximal embolization and improvement in platelet counts at 3 and 7 days. The manuscript is well written and the data are well presented.

Key Points:

Methods: Please explain in more detail the use of the mixed linear regression model (fixed and random effects). Why do the authors believe this model is superior to the nonparametric comparison of laboratory values at time 0 and time x using the signed-rank test?

Treatment outcomes can be analyzed using only a final measurement like a Wilcoxon signed-rank test.

Unfortunately, much of the information captured with repeated measurements, such as the pattern of outcomes across the timepoints, is not assessed. Another weakness of the signed-rank test is its limited power. As observations are converted to ranks and only ranks are used in the test statistic, the signed-rank test does not use all available information in the original data, leading to lower power when compared with tests that use all data. Furthermore, missing values in the data set lead to the exclusion of the pair. Nonetheless, for descriptive data, we used the signed rank test to compare median baseline values and values at the end of the observation period of 30 days. We deemed it best to provide an outline of the results with a test that is familiar to most readers and more comprehensible than the models discussed below.

Linear Mixed Models (LMM) account for correlations between repeated measurements within each patient and are well suited to settings in which the individual trajectory of a particular outcome for a study participant over time is influenced both by factors that can be assumed to be the same across a collective, so-called fixed effects (such as the effect of an intervention), and by factors that are likely to vary from patient to patient, so-called random effects. The results reported after 30 days show the impact of the intervention on the trajectory of the patient across the different time points and do not only compare the first and the last data point as e.g. a signed-rank test. Repeated measures analysis of variance (rmANOVA) e.g., which is often used for analyzing longitudinal data, does not have the same flexibility as LMM and can yield misleading results due to its more rigid assumptions (e.g. all effects are considered fixed). Another advantage of mixed modeling over rmANOVAS is the handling of missing data. Under the “missing-at-random” assumption, mixed models can accommodate unbalanced data patterns and use all available data points and patients in the analysis, resulting in a more powerful study.

To conclude, we believed the advantages of mixed models over e.g. a signed rank test and rmANOVAs to be the additional information inferred from the trajectory of an individual patient across different time points, the flexibility of model building in terms of fixed and random effects and the superior manageability of missing data and statistical power.

We totally agree with you that a signed-rank test and LMM, under certain circumstances, shed light on the same question from different angles which might be confusing for readers. We went into greater detail of our model building approach in the methods section and restructured the results section accordingly. If you still find the use of to different test statistics confusing for potential readers, we would report solely the results of our longitudinal mixed model analyses.

Figure 2: To better assess the change in transaminase levels after proximal versus distal embolization, I propose to plot median delta transaminase levels (compared with day 0) for all time points.

We felt the reporting of median delta values might cause confusion as e.g. bilirubin, thrombocytes and transaminases distribute across entirely different ranges, depending on the respective units. Defining the baseline as 1 (X0) and plotting all following time points as delta (X1-X0) would make the graphs quite confusing. Removing the baseline (defined as 1) from the visualization for the sake of clarity, we would deny the reader an important piece of information. So, we struggle to implement your suggestion without adding complexity or truncating the information. We adjusted all measurements for baseline values for the linear mixed model analyses and the visualization with forest plots though.

Do the authors have data on white blood cell counts or CRP levels after embolization to assess whether systemic inflammation differs between proximal and distal embolization?

Unfortunately, we obtained no data on systemic inflammation as we focused primarily on surrogate parameters of liver function.

The increase in GPT levels at 3 days alongside similar levels at 30 days could indicate transient liver hypoperfusion after the procedure but a similar long-term outcome. Please discuss.

Thank you very much for this helpful remark. We expanded on this in the discussion.

Minor points:

The authors state that there were no procedure-related complications. Does this apply to pain, splenic abscesses, and infections?

No splenic abscess or infection was seen. Periinterventional pain was not documented. Unfortunately, there is no accessible documentation on post-interventional pain on the ward. We added this in the Results section.

Were patients routinely treated with nonsteroidal anti-inflammatory drugs, steroids, or antibiotic prophylaxis?

After OLT all patients received a calcineurin-inhibitor-based immunosuppressive protocol and steroids which are usually tapered within the first month after surgery. NSAR or antibiotics are not part of our standard of care for SA embolization but may have been administered at discretion of the referring department depending on the clinical scenario. We added this in the methods section.

Please provide any other characteristics of the study cohort, including time since transplant and immunosuppression, that might affect transaminase progression.

Thank you very much, we included the time to treatment in table 1 (patient characteristics). Unfortunately, we are not able to restore all information on immunosuppression in detail and consistently. Yet, we believe this could in fact be subject to further scientific evaluation.

Was hepatic artery stenosis excluded in all patients?

One patient underwent stenting of the hepatic artery for a dissection but was not included for statistical analyses. Stenosis was excluded in all other patients. Any post-transplant patient is routinely screened with doppler ultrasound for vascular pathologies on a daily routine for at least one week after surgery and is referred to our department in case of findings consistent with e.g. hepatic artery stenosis.

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28 Jan 2022

Splenic artery steal syndrome in patients with orthotopic liver transplant: where to embolize the splenic artery?

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15 Feb 2022

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Splenic artery steal syndrome in patients with orthotopic liver transplant: where to embolize the splenic artery?

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