Moderately Macrosteatotic Livers Have Acceptable Long-Term Outcomes but Higher Risk of Immediate Mortality

Abstract

Background and aims.

Liver transplantation is the most effective treatment for end-stage liver disease (ESLD). Whether moderately macrosteatotic livers (30%–60%) represent a risk for worsened graft function is controversial. The uncertainty, in large part, is owing to the heterogeneous steatosis grading. Our aim was to determine the short- and long-term outcomes of moderately macrosteatotic allografts that were graded according to a standardized institutional protocol.

Methods.

We performed a retrospective analysis of transplants performed between 1994 and 2014. All patients with allografts biopsied pretransplantation were included. Relevant donor and recipient variable were recorded. Moderately macrosteatotic livers were compared with mildly macrosteatotic and nonsteatotic livers. Primary outcomes of interest were patient survival at 90 days, 1 year, and 5 years. Cox regression analyses were carried out to compare survival between the 2 groups.

Results.

We compared 65 allografts with moderate macrosteatosis and 810 with no or mild macrosteatosis. Patients with moderately macrosteatotic allografts were 2.69 times as likely to die within the first 90 days after transplant (75.1% vs 91.6% survival) after adjusting for donor age, donor race, recipient age, recipient race, recipient body mass index, recipient diabetes, presence of hepatocellular carcinoma, days on waitlist, Model for End-Stage Liver Disease (MELD) score at transplantation, cold ischemia time. However, for recipients who survive 90 days, moderately macrosteatotic allografts had comparable long-term survival.

Conclusion.

Our study shows that moderate macrosteatosis is a strong predictor of early but not late mortality. Further studies are needed to distinguish the specific cohort of patients for whom moderately macrosteatotic allografts will lead to acceptable outcomes.

Liver transplantation is the most effective treatment for end stage liver disease (ESLD). The rising incidence of ESLD [1] secondary to an increase in fatty liver disease [2] has resulted in a growing organ scarcity, and, consequently, higher death rates among those awaiting transplantation. To augment the donor pool, some transplant centers [3] have adopted the use of “extended-criteria donors” (ECD) for liver allografts. Among the different ECD grafts, hepatic steatosis is one of the most common findings. Steatosis is divided into microvesicular and macrovesicular (MaS) [4]. Analysis of ECD donors has identified macrovesicular steatosis [5–7], increased donor age [5,6], and prolonged cold ischemia time (CIT) [5,6] as risk factors for delayed graft function and primary nonfunction.

Although outcomes of allografts with microvesicular steatosis are similar to those without steatosis [8], the degree of MaS correlates with the risk of delayed graft function and primary nonfunction [9,10]. Function and survival of allografts with MaS, which can be further divided based on percent steatosis (i.e., mild: < 30%; moderate: 30%–60%; severe: > 60%), are highly variable. Although some investigations conclude that moderate MaS is an independent risk factor for lower graft survival [11,12] and poor early graft function [13,14], others have reported that this risk might be mitigated with optimization of other factors such as cold ischemia time [11].

Inconsistencies in donor liver biopsy assessment resulting from interobserver variability, sampling error [15], pathologist availability [16], and heterogeneity in pathologist expertise pose major challenges with regard to organ utilization decision-making [17]. Although standardizing steatosis classification remains an active area of research [18–20], many of the prior studies of MaS graft outcomes relied on biopsy data that may have confounded the ultimate findings [7]. It is therefore important to evaluate the impact of moderate MaS on recipient outcomes when all biopsies are evaluated at a single institution by expert histopathologists. The objective of this study was to analyze liver transplantation outcomes and pretransplant biopsy data from our institution and determine the impact of moderate MaS on patient and allograft survival. We hypothesized that recipients of liver allografts with moderate MaS can have acceptable posttransplantation outcomes.

MATERIAL AND METHODS

Study Design and Patient Population

We conducted a retrospective analysis using the medical records of patients receiving liver transplants at UPMC between January 2, 1994, and March 8, 2014. This study was approved by the University of Pittsburgh Institutional Review Board under protocol number PRO13060220 and is in compliance with the ethical standards set by the Helsinki Congress. Patients with allografts biopsied pretransplantation were included. Retransplants, living donor transplants, and patients with incomplete clinical data were excluded. Variables of interest included donor age, sex, race, donor type (e.g., donation after brain death vs donation after cardiac death), CIT, and the presence of diabetes mellitus and/or hypertension. Abstracted recipient data included age, sex, body mass index, race, presence of hypertension, diabetes, history of hepatocellular carcinoma (HCC), Model for End-Stage Liver Disease (MELD) score at transplantation, and days on waitlist.

Outcomes

Our primary outcome was patient survival at 90 days, 1 year, and 5 years after transplantation. A secondary outcome included organ survival, defined as time between transplantation and patient death or retransplant.

Statistical Analysis

The χ² test and Kruskal-Wallis tests were used to compare group differences in recipient, donor, and transplant characteristics. Ninety-day, 1-year and 5-year survival rates were compared using the Kaplan-Meier method [21], and median survival times were calculated for allografts with no or mild MaS (<30% MaS) and moderate MaS (>30% MaS). Owing to a violation of the proportional hazard assumption, Cox regression could not be completed over the entire follow-up period. Instead, Cox regression analyses were carried out in 2 separate models (before and after 90 days). After assessing that the assumptions of the Cox model were met, proportional hazard model analyses were performed to compare the short-term (death before 90 days) and long-term (death after 90 days) survival between the 2 groups. The regression model included recipient age, recipient body mass index, race (White vs non-White), recipient diabetes, history of HCC, waitlist days, meld at transplant, CIT, donor age, and donor race (white). All statistical analysis was performed using SAS software version 9.4 (Cary, NC, USA).

Pathology Assessment

At our institution, a pretransplant biopsy was based on the presence of clinical and visual characteristics associated with increased risk, such as age, donation after cardiac death, CIT, and visualization of steatosis at procurement, but ultimately the decision rests on the clinical judgment of the transplant surgeon. The same group of pathologists at our institution, specialized in transplantation, who first recognized the risk of dysfunction after transplantation of fatty donor livers [22] evaluated donor livers by frozen section examination. The final pathologic evaluation of the pretransplant biopsy was always performed by the pathologist group at our institution, including for donors located at outside facilities. Steatosis was semiquantitatively evaluated to the nearest 10% using hematoxylin-eosin stains of 5-micron thick frozen sections from 1 needle core (16 gauge) from both right and left lobes and a right lobe subcapsular wedge. Microvesicular steatosis was identified as small vacuoles that occupied < one-third of the shortest nuclear diameter of diploid-appearing hepatocytes that did not displace the nucleus to the cellular periphery. Macrovesicular steatosis was defined as larger globules that are visible at 20 × magnification, which is generally greater than the nuclear diameter that displaces the nucleus toward the periphery (Fig 1). Tissues were then processed, and permanent sections were prepared and stained for routine examination. The frozen sections were then compared to Hematoxylin-eosin–stained permanent section evaluation. This was accomplished with a weekly quality assurance conference review where significant discrepancies between frozen and permanent section diagnoses were identified.

Fig 1.

Hematoxylin-eosin (H&E-2X) stained preimplantation donor frozen sections biopsies with macrosteatosis estimated to involve 10% (A), 30% (B), 40% (C), and 60% (D) of lobular area.

Two approaches have been used to maximize agreement in macrovesicular steatosis scoring. First, a diagram with different percentages of steatosis was created to assist semiquantitative assessment of steatosis (Fig 2). Second, cases showing disagreements of >15% in macrovesicular steatosis assessment were reviewed and discussed at the Quality Assurance conferences.

Fig 2.

Standardized diagram illustrating the severity of macrovesicular steatosis used to improve accuracy of estimating the percentage of macrovesicular steatosis. (Published in Busuttil and Klintmalm [23]).

RESULTS

Of the 3287 liver transplants performed at the study institution between January 2, 1994, and March 8, 2014, 810 allografts with <30% MaS and 65 allografts with >30% MaS were transplanted. Donor and recipient characteristics are demonstrated in Table 1. In total, 281 of the 810 (34.7%) ≤30% MaS allografts had no MaS and the remaining 529 had mild (1%–30%) MaS. The survival of those who received a liver with <30% MaS was 91.6%, 83.5%, and 66.2% at 90 days, 1 year, and 5 years, respectively. In comparison, survival with >30% MaS was 75.1%, 70.4%, and 52.0%, respectively. Kaplan-Meier curves demonstrate an early decline in survival for the >30% MaS group compared with the <30% MaS group but had comparable long-term survival (Fig. 3A, 4A). The posttransplantation laboratory values of aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin, and international normalized ratio (INR) were compared between the 2 groups (Table 2). We found that ALT and AST levels were significantly higher in the ≥30% MaS group than in the ≤30% MaS group on postoperative days 1 and 3, total bilirubin level was higher on postoperative day 7, and INR was higher on postoperative day 1. Time on transplant waitlist (hazard ratio [HR], 1.004; 95% confidence interval [CI], 1.001–1.006; P = .003), CIT (HR, 1.001; 95% CI, 1.000–1.002; P = .042), and MELD at transplant (HR, 1.058; 95% CI, 1.032–1.085; P = < .0001) were all significantly associated with 90-day mortality among the entire patient cohort. Notably, receiving a >30% MaS allograft was a significant independent risk factor for mortality within 90 days (HR, 2.687; 95% CI, 1.47–4.90) after adjusting for donor demographics, recipient demographics and comorbidities, time-to-transplant, MELD, and CIT. However, this risk of death is similar between <30% MaS and >30% MaS groups after 90 days (Fig 3B). There were no statistically significant differences in baseline characteristics between those patients with >30% MaS allografts that died within 90 days (n = 16) and those that survived (n = 48) but several trends emerged (Table 3). First, patients who died within 90 days had been on the waitlist longer, 538.7 vs 237.1 days, they were more likely to be male (75% vs 47.9%), non-White patients (12.5% vs 2.1%), and they had a lower prevalence of HCC (6.7% vs 16.7%).

Table 1.

Donor and Recipient Characteristics of Liver Transplants With No or Mild Macrosteatosis (<30% MaS) and Moderate Macrosteatosis (>30% MaS)

Donor Characteristic	>30% MaS (n = 65)	<30% MaS (n = 810)	P Value
Age, y	48.9 ± 15.1	52.5 ± 17.2	.097
Sex, male	35 (53.9%)	446 (55.1%)	.85
Race, white	63 (96.9%)	702 (86.7%)	.017*
BMI	32.8 ± 8.6	27.56 ± 7.2	< .0001*
Diabetes	6 (9.4%)	84 (10.4%)	.86
Hypertension	30 (46.9%)	368 (45.4%)	.84
CIT, min	710.4 ± 203.1	676.5 ± 178.2	.15
Donor Type			.98
DBD	60 (92.3%)	747 (92.2%)
DCD	5 (7.7%)	63 (7.8%)
Recipients Characteristics
Age, y	54.6 ± 10.4	54.5 ± 10.1	.86
Sex, male	44 (67.7%)	523 (64.6%)	.62
Race, white	63 (96.9%)	784 (96.8%)	.950
BMI	29.3 ± 5.8	28.8 ± 5.9	.476
Diabetes	5 (7.8%)	148 (18.3%)	.030*
Hypertension	14 (21.9%)	190 (23.5%)	.770
HCC	9 (14.1%)	92 (11.4%)	.530
Days on waitlist	84 (18–293)	77 (21–211)	.097
MELD at transplant	16.2 ± 6.7	17.3 ± 8.1	.310

Continuous variables are presented as mean ± SD or median (IQR). Categorical variables are presented as n (%).

BMI, body mass index; CIT, cold ischemia time; DBD, donation after brain death; DCD, donation after cardiac death; HCC, hepatocellular carcinoma; MELD, Model for End-Stage Liver Disease score; SD, standard deviation.

^*P-value <0.05

Fig 3.

Survival of recipients receiving liver transplants with <30% MaS and >30% MaS and hazard (mortality) rate over 5000 days posttransplant. (A) A Kaplan-Meier plot depicts <30% MaS (blue) and >30% MaS (red) liver transplant recipient survival over 5000 days. Vertical gray line signifies 90-day timepoint. (B) The estimated hazard rate for the 2 groups over the same period is shown. Hazard is defined as death on the corresponding day. MaS, macrosteatosis.

Fig 4.

Graft survival rates for livers with <30% MaS and >30% MaS and graft failure rate over 5000 days posttransplant. (A) Kaplan-Meier plot depicts of < 30% MaS (blue) and > 30% MaS (red) liver transplant recipient survival over 5000 days. Vertical gray line signifies 90-day timepoint. (B) The estimated hazard rate for the 2 groups over the same period is shown. Hazard is defined as death on the corresponding day. MaS, macrosteatosis.

Table 2.

Posttransplant Laboratory Values of Liver Transplants With No or Mild Macrosteatosis (<30% MaS) and Moderate Macrosteatosis (>30% MaS)

	>30% MaS (n = 65)	<30% MaS (n = 810)	P Value
AST
POD#1	2949 (1862–4857)	1284 (670–2599)	< .001*
POD#3	691 (343–1241)	463 (229–925)	.011*
POD#7	48 (33–77)	46 (30–70)	.31
POD#30	30 (23–50)	31 (21–55)	.93
ALT
POD#1	876 (598–1291)	589 (318–1048)	.002*
POD#3	566 (368–954)	472 (257–902)	.043*
POD#7	109 (72–157)	107 (70–172)	.60
POD#30	42 (30–66)	42 (26–73)	.95
Total Bilirubin
POD#1	4.6 (3.0–6.6)	4.5 (2.8–6.9)	.80
POD#3	4.1 (2.2–7.0)	3.2 (1.8–6.0)	.042*
POD#7	7.2 (3.7–11.8)	4.3 (2.1–9.1)	< .001*
POD#30	1.4 (1.0–3.8)	1.3 (0.8–2.9)	.15
INR
POD#1	1.9 (1.6–2.2)	1.7 (1.5–2.1)	.049*
POD#3	1.3 (1.2–1.7)	1.3 (1.1–1.5)	.077
POD#7	1.2 (1.1–1.3)	1.2 (1.1–1.3)	.28
POD#30	1.2 (1.1–1.3)	1.20 (1.1–1.3)	.68

Continuous variables are presented as median (IQR).

AST, aspartate transaminase; ALT, alanine transaminase; INR, international normalized ratio; POD, postoperative day.

^*P-value <0.05

Table 3.

Donor and Recipient Characteristics of >30% MaS Liver Transplants Who Survived Less Than 90 Days (<90 d) and More Than 90 Days (>90 d)

Donor Characteristics	<90 d (n = 16)	>90 d (n = 49)	P value
Age, y	49.9 ± 16.1	49.0 ± 14.6	.83
Sex, male	12 (75.0%)	23 (47.9%)	.06
Race, White	14 (87.5%)	48 (97.9%)	.15
BMI	32.4 ± 9.0	32.9 ± 8.6	.67
Diabetes	2 (13.3%)	4 (8.3%)	.62
Hypertension	7 (43.8%)	22 (45.8%)	.95
CIT, Min	645 ± 223.3	733.9 ± 195.1	.14
Donor Type			.32
DBD	16 (100.0%)	44 (89.6%)
DCD	0 (0.0%)	5 (10.4%)
Recipients Characteristics
Age, y	53.38 ± 13.11	55.35 ± 9.25	.51
Sex, male	11 (68.8%)	32 (66.7%)	1
Race, white	13 (81.3%)	47 (95.8%)	.095
BMI	28.8± 6.26	29.5 ± 5.80	.69
Diabetes	1 (6.7%)	4 (8.3%)	1
Hypertension	2 (12.5%)	12 (24.5%)	.48
HCC	1 (6.6%)	8 (16.7%)	.67
Days on waitlist	538.7 ± 974.6	237.1 ± 431.1	.26
MELD at transplant	16.67 ± 8.86	16.06 ± 6.10	.77

Continuous variables are presented as mean ± SD. Categorical variables are presented as n (%).

BMI, body mass index; CIT, cold ischemia time; DBD, donation after brain death; DCD, donation after cardiac death; HCC, hepatocellular carcinoma; MELD, Model for End-Stage Liver Disease score; SD, standard deviation.

DISCUSSION

Our results demonstrate that patients who receive liver allografts with moderate MaS have higher short-term mortality but acceptable outcomes once they are out of the immediate postoperative period. To the authors’ knowledge, this is the largest series to date documenting the natural history of allografts with ≥30% MaS that were uniformly graded by a single group of pathologists. Among recipients of moderately MaS liver allografts, those who had early mortality tended to have more days on waitlist, are non-White men, and have lower prevalence of HCC than those who survived, but these differences were not statistically significant.

Donor liver macrosteatosis is an important risk factor for graft dysfunction and poor outcome after liver transplantation. The worsening global burden of non-alcoholic fatty liver disease (NAFLD) [2] necessitates that a larger proportion of potential donors will have a degree of fatty liver disease. The use of moderately MaS allografts is controversial given limited clinical experience and concern for poor graft function. Existing literature has found outcomes ranging from consistently poor [13–22,24] to acceptable in certain clinical scenarios [7,9]. Heterogenous grading of donor liver steatosis compounds these challenges and makes interpretation of the available data difficult. Quantification of liver steatosis has been criticized as being highly observer dependent, poorly reproducible, and not consistent with software-assisted quantitative morphometry [17].

Consequently, it is imperative that methods of classification and quantification of steatosis become standardized. As a first step in addressing this, the current study evaluates short- and long-term outcomes for patients receiving moderately MaS liver allografts based on a homogenous grading methodology. The current steatosis grading was performed by expert transplant histopathologists at a single institution using standardized diagrams and quality assurance reviews (see Methods), which allows for a more cogent analysis. We found that recipients of allografts with moderate MaS have significantly worse 90-day survival compared to grafts with no or mild MaS. While controlling for confounding factors, patients who received livers with moderate MaS are 2.69 times as likely to die within 90 days (HR of 2.69, 95% CI 1.47–4.90). This finding largely corroborates results of prior studies that demonstrate poor early outcomes of steatotic grafts [13,25].

Interestingly, patients with moderately MaS allografts who survived the first 90 days did not have significantly worse outcomes than their counterparts with allografts with no to mild MaS. With a follow-up time of almost 14 years (exactly 5000 days) used in our study, the survival of >30 vs <30 shows no divergence after 90 days. The patients who experienced early mortality had more days on waitlist, were more likely to be non-White men, and had lower prevalence of HCC. Although these differences were not statistically significant, minimizing these factors may improve outcomes in patients who receive moderately MaS liver allograft.

Increased susceptibility of a steatotic graft to ischemia reperfusion injury is one proposed mechanism for early mortality difference [26]. We found significantly higher AST, ALT, total bilirubin, and INR levels during the posttransplantation period following moderately MaS allograft transplantation. This supports the hypothesis that these organs do get worse following ischemia reperfusion, in particular because the CIT were similar between our groups, but importantly there were no differences in survival beyond 90 days. Our data suggest that patients with >30% MaS allografts have equal long-term survival than their counterparts with <30% MaS livers. Indeed, a recent study demonstrated full resolution of donor NAFLD after transplantation [27], pointing to a potential explanation for our long-term survival. Whether resolution of donor NAFLD leads to better long-term recipient survival is an important clinical question. Further studies are needed to investigate this association and elucidate the mechanisms behind it because they may provide insights and therapies to help the growing NAFLD patient population.

The reliability of our findings is supported by the expertise of the transplant pathologists who evaluated the pretransplantation biopsies. Training in methodology in identification of MaS livers yields more reliable data in comparison to that generated by pathologists with variable training experience. Inter-rater reliability mitigates heterogeneity in steatosis assessment, which may confound large-scale analyzes that rely on aggregate data from multiple centers.

In our center, a significant (>15%) discrepancy between the frozen and permanent sections of macrovesicular steatosis was noted in <5% (3.1%) of cases. In the majority of these cases, the discrepancy was secondary to an overestimation of the steatosis level on the frozen section compared to the paraffin-embedded tissue.

We acknowledge that the retrospective study design leads to several limitations on our analysis. First, given the broad timeframe of this study, electronic medical records for patient data from early periods may have been incomplete with regard to pertinent technical details of the transplant itself. Additionally, our sample size of allografts with moderate MaS was small and may have resulted in analyses underpowered to predict risk factors for 90-day survival. Also, comparing only the liver allografts that were biopsied may introduce a selection bias because even the allografts found to have no macrosteatosis may, in theory, represent a cohort of patients at higher risk for worse outcomes than the nonbiopsied counterparts given the initial concerns that led to the decision to biopsy. However, the strength of this study is in the uniformity of the pretransplant biopsy grading and the long-term outcome data.

CONCLUSIONS

In summary, we report our experience with transplantation of moderately macrosteatotic liver allografts. Patients who receive these organs had a higher 90-day mortality when compared to those receiving livers with no or only mild MaS. This difference remained even after controlling for several potential confounding factors. However, and importantly, if recipient of grafts with moderate MaS survives the first 90 days, their risk of mortality was equal to those with no or mild MaS beyond 90 days. This has important implications in the ECD selection/allocation. Further efforts are needed to better elucidate the underlying causes or donor/recipient factors associated with early mortality that can be used to select the appropriate subset of patients best suited to receive ≥30 MaS livers. In addition, all pretransplantation biopsies were assessed by members of a single, high-volume pathology department, with a standardized grading system making this a uniformly graded cohort, which helps to mitigate the biopsy grading variability that is known to be a major limitation in this field. Our findings highlight the need for future studies to further this work in identifying those patients in whom moderately MaS allografts could yield consistently acceptable outcomes.