Hepatic Steatosis in Organ Donors: Disparity Between Surgery and Histology? J.W. Rey, U. Wirges, H.P. Dienes, and J.W.U. Fries ABSTRACT Background. In times of organ shortage, use of marginal cadaveric livers has become increasingly important to reduce pressing organ demand and rising death rates while awaiting donations. Indisputably, fatty change in donor livers is a risk factor for poor initial function after orthotopic transplantation. However, identifying and rejecting marginal from good donor livers is one of the most difficult surgical tasks. Unfortunately, a liver biopsy with rapid histological diagnosis is rarely performed to identify marginal livers. Methods. From 2005 to 2008, we investigated 36 livers of organ donors, which were explanted but not transplanted or underwent liver wedge biopsy during organ donation. All livers underwent standard surgical procedures and were allocated by Eurotransplant International Foundation. After unsuccessful allocation, explanted livers were photographically documented, formalin-fixed, and analyzed histopathologically. Results. Seven livers were classified as good organ quality by the surgeon (19.4%); 15 were acceptable (41.6%); and 14 poor (39%). In 63.8% of livers, a frozen section was performed; 6/36 cases (16.7%) showed macrovesicular and microvesicular steatosis of less than 30%. In addition, all six cases fulfilled two or less extended donor criteria, as defined by the German Medical Association. Conclusion. More marginal livers from cadaveric organ donors could have been transplanted. To extend the transplant pool of liver grafts, liver biopsies should be performed in all cases of acceptable and poor livers. If frozen section analysis is performed, a wedge liver biopsy should be taken from at least two different segments of the liver to validate the histological results. ATTY LIVER DISEASE is an increasing problem in Western countries. The prevalence of hepatic steatosis is approximately 20% in industrial nations.1 Therefore, hepatic steatosis is a commonly noticed, most prevalent condition among donated liver grafts. The pressing demand for organs and increased patient death rates while awaiting organ transplantation have led to the use of cadaveric livers with hepatic steatosis for transplantation. For a successful evaluation, reliable and objective means are needed to assess the liver before transplantation. The severity of steatosis is traditionally classified as mild (⬍30%), moderate (30%– 60%), or severe (⬎60%).2 Hepatic steatosis describes two major histological patterns following fat accumulation within hepatocytes. Macrovesicular steatosis (MaS) is histologically described as a single fat vacuole within an individual hepatocyte that symptomatically squeezes the nucleus to the periphery. The most
F
associated disorders of MaS are reported to be alcohol abuse, obesity, diabetes mellitus, and dyslipidemia. MaS can occur with microvesicular steatosis (MiS). MiS is associated with toxins, metabolic disorders, Reye’s syndrome, sepsis, or intensive care treatment. It is characterized by an accumulation in the cytoplasm of one to several minute vacuoli that are smaller than the nucleus.3,4 Many authors have reported that MiS was not directly related to patient survival after transplantation.4,5 With the exception of a From the Institute of Pathology, University Hospital of Cologne, Germany, and German Organ Procurement Organisation (DSO), Essen, Germany. Address reprint requests to Dr. Johannes W. Rey, University Medical Center Mainz, Department of Internal Medicine I, Langenbeckstraße 1, 55131 Mainz, Germany. E-mail: johannes.
[email protected]
© 2009 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.06.121
Transplantation Proceedings, 41, 2557–2560 (2009)
2557
2558
toxin etiology, fatty liver changes can be totally reversible. However, fatty liver disease can evolve from parenchymal inflammation to fibrosis or even cirrhosis. Indisputably, fatty change in donor livers is a risk factor for poor initial function after allogenic transplantation. Especially, greater than 30% MaS in a cadaveric liver graft combined with prolonged cold ischemia and poor recipient health, is associated with initially poor graft function.6,7 Identifying marginal from good donor livers is one of the most difficult surgical tasks. Thus, surgical experience and evaluation criteria have increased importance in liver transplantation. Visual inspection and palpation are the most commonly used methods for surgeons to establish liver quality. The positive predictive value of a surgical appraisal of liver steatosis has been reported to be 71% for severe; 46% for moderate; and 17% for mild steatosis.2 Certainly, severe steatosis can be identified by yellow discoloration after perfusion and rounded edges. This subjective test is less sensitive with mild to moderate steatosis. Unfortunately, a liver biopsy and rapid histological diagnosis is rarely performed to identify marginal livers. The most common feature to evaluate liver quality worldwide is microscopic examination of a wedge biopsy obtained during organ procurement. When donor surgeons suspect steatosis by inspection, a frozen section is only performed in 38% to 47% of cases.8 Due to time restraints during the transplantation process, frozen sections are preferably performed, with hematoxylin and eosin (H&E) staining most common. Alternative methods to detect steatosis with high sensitivity are Sudan-III staining, toluidine blue staining, and oil red O staining. Although oil red O staining is sensitive to detect steatosis, it is highly dependent on technical expertise, which is most often not available during the night hours. In addition, it requires a processing time that exceeds the allocation process.8,9 Standardization of biopsy procedures and the weight of frozen section liver biopsy results with reference to the surgeon’s evaluation of liver grafts are the subjects of this report. Thus, we evaluated 36 explanted but subsequently not transplanted liver specimens and their respective frozen sections. We correlated all clinical, laboratory, macroscopic, and histological data to evaluate reasons for the rejection of the organ and to identify potential problems in the evaluation process. These data demonstrated the need for more frequent use of histological evaluations by frozen sections. We noted that specimens from at least two different sites (right and left liver lobe)10 were necessary to provide valuable evaluation criteria for livers of questionable transplantation quality upon surgical evaluation. MATERIALS AND METHODS From 2005 to 2008, we investigated 36 organ donor livers, which were explanted but not transplanted. After brain death was determined according to the guidelines of the German Medical Association and permission obtained, individuals were selected as potential organ donors. All livers were allocated by the Eurotransplant International Foundation. Organ donation was performed by local
REY, WIRGES, DIENES ET AL surgeons experienced in at least 10 organ donations as required. Explantation of the liver was performed via a median laparatomy using perfusion with histidine-tryptophan-ketoglutarate solution (Custodiol) or University of Wisconsin solution (Viaspan). If the transplant surgeon required a frozen section, a wedge biopsy of 1-cm side length was taken (from segment 3 and/or 6). The wedge was split lengthwise; one half was snap frozen in liquid nitrogen from which 4-m-thick sections were cut, briefly fixed in 4% buffered formalin (pH 7.4), rinsed, and counterstained with H&E. After dehydration in rising concentrations of alcohol, the coverslipped section was analyzed by routine light microscopy. The remaining half of the wedge biopsy was directly fixed in buffered formalin overnight. After paraffin embedding, 3 to 4-m-thick sections were cut. Routinely used staining solutions (besides H&E) included Gomori’s stain, van Giesson elastic stain, and iron staining using Berlin blue. The frozen section material was postfixed overnight and also stained as described for the directly fixed biopsy tissue. If the allocation was unsuccessful, nontransplanted livers were approved for histopathologic evaluation. Explanted livers were photographically documented, formalin-fixed, and histopathologically analyzed11 as described for the directly fixed part of the wedge biopsy. However, a wedge was taken from each liver section to enable comparison of potential morphological differences. Histological classification was performed according to the criteria published by Kleiner et al,11 which analyzes steatosis, inflammation, fibrosis, and liver cell damage.
RESULTS Clinical, Laboratory, and Pretransplant Evaluation
The workup and subsequent evaluation of available clinical data of the 36 liver explants in this study revealed that the average age of the potential donors was 61 (range ⫽ 28 – 82) years. Of these donors, 21 were males and 15 females. Median body mass index was 26.1 kg/m2. Cause of death was primary cerebral damage in 75%, or secondary cerebral damage in 25%, leading to intensive care unit hospitalization for an average of 4.9 days. Potential organ donors displayed on average 2.9 extended donor criteria (EDC) as defined by the German Medical Association, and 13.2% of patients had diabetes. Alcohol abuse ranging from mild to severe was documented in 26.3%; 8/36 donors (21%) were tested exclusively positive for anti-HBc. In 77% of liver procurements, organ perfusion was performed using on average 10 liters of Custodiol solution; 33% were preserved by an average of 6.5 liters of Viaspan. In total, 7 livers showed good organ quality as classified at surgery (19.4%), 15 were acceptable (41.6%), and 14 of poor quality (39%). Liver Steatosis
In 47.2% (17/36) of cases, steatosis was noticed preoperatively by sonography. However, histology did not support this finding in 29.4% of cases. In contrast, the sonographically based prediction was falsely negative in 35.7% of all cases; 23/36 cases (63.9%) showed less than 50% MaS or MiS. During liver procurement, frozen sections were performed in 23 cases. They showed complete agreement in the degree of steatosis with paraffinized material (evaluated independently by two different pathologists) in 20 cases (87%). Disagreement about the degree of steatosis by more
HEPATIC STEATOSIS IN ORGAN DONORS
2559
strating that an equal number of livers with clearly different degrees of steatosis (mild vs severe) presented macroscopically with nearly identical (yellow) coloring. Among the 36 livers, 30.5% (n ⫽ 11) revealed a maximal degree of steatosis of 30% without signs of fibrosis or cirrhosis. They showed negative virological evaluation, and maximal 2 EDC. Among those, five cases had nonallocation of the organ for reasons independent of donor criteria.
25%
20%
15%
mild
moderate
Liver Fibrosis
yellow
yellow-brown
brown
yellow
0%
yellow
5%
yellow-brown
10%
severe
Fig 1. Association between liver color and degree of steatosis. Figure displays the percentage of livers with different degrees of steatosis (mild, moderate, severe) depending on liver color (yellow, yellow-brown, and brown-red) after organ perfusion.
than 10% between frozen versus permanent sections occurred in three cases: two MiS and one MaS. By correlating photographic images of livers with their respective histopathologic changes, we noticed individual cases of unexpected discrepancies between liver color and morphological degree of steatosis, as highlighted in Fig 2. Thus, it was possible to observe various degrees of steatosis (mild vs moderate or severe) in livers with indistinguishable (particularly yellowish) color intensity. Based on this result, we correlated all photographically livers similarly. This evaluation is shown in Fig 1, demon-
A
B
Among the 36 evaluated organs, liver fibrosis or cirrhosis was detected in 22 (61%). In 13 (36%), there was mild to moderate fibrosis (grade 1); three of which showed bridging fibrosis (grade 3) or complete cirrhotic changes (grade 4). The remaining 12 livers (39%) had no detectable fibrosis. Analyzing cases with fibrosis further, we observed that some had specific morphological changes. Among those was a case with diffuse but exclusive capsular and focal subcapsular fibrosis and another case with fibrosis restricted to a single liver segment (segment 7), most likely due to a regional artery occlusion. DISCUSSION
A successful evaluation of a liver graft for transplantation is based on several essential criteria: donor medical history, laboratory data on liver function, virological analysis, sonography, as well as intraoperative liver exploration particular for color and palpation. According to our study, the subjective criteria of color and palpation crucially depend on the experience of the explanting surgeon.
C
D
Fig 2. Comparison between macroscopical appearance and coloring versus degree of morphological steatosis. Both livers (A and B) photographed with f5.6, time 1/80 seconds and ISO 200 with indirect TTL flashlight. Histopathologically, liver (A) shows a moderate to severe degree of steatosis (C), while in liver (B) the degree of steatosis is mild (D). The presented light microscopic picture reflects a degree of change characteristic for all liver segments.
2560
The strength of the macroscopic surgical evaluation lies in the recognition of liver tumors, progressive fibrosis and cirrhosis as well as vascular variations and hepatic vein thrombosis. The surgical evaluation of fat accumulation and early degrees of fibrosis by macroscopic examination, however, has a low predictive value, according to our results. Particularly yellowish color changes can be misleading, resulting in unwarranted rejection of a liver transplant. Thus, our data indicated the need for more frequent histopathologic evaluation by frozen sections to decrease the number of nontransplantable livers among questionable cases. With this goal in mind, a wedge biopsy of 1-cm side length should be performed in at least two liver segments, one for each liver lobe (ie, segments 3 and 6 or 7) as suggested previously by Frankel et al.10 Looking at the potential gain of information by a biopsy, we have suggested that all livers to be transplanted should be biopsied. Our analysis, utilizing retrospective available donor data, pointed to the possibility that 16.7% (6/36) of all nontransplanted livers might have been transplantable; this would have helped to reduce the pressing organ demand. Continuously collecting clinical, surgical, and histopathologic data in a central registry of all transplanted livers may provide valuable information as to whether the evaluation at time of transplantation was correct. If a posttransplant biopsy is taken after liver transplantation, the results of its histopathologic evaluation should likewise be collected in the same registry. This measure will improve our knowledge of the importance of morphological alterations and their significance at the time of transplantation. ACKNOWLEDGMENTS We gratefully acknowledge the fruitful cooperation with all visceral transplant surgeons from the University Hospitals in North Rhine-
REY, WIRGES, DIENES ET AL Westphalia, particularly from Bochum, Bonn, Essen, Münster, and Köln. Additionally, we thank all transplant coordinators from the German Organ Procurement Organisation (Deutsche Stiftung Organtransplantation, DSO) in the region of North Rhine-Westphalia.
REFERENCES 1. Neuschwander-Tetri BA: Nonalcoholic steatohepatitis and the metabolic syndrome. Am J Med Sci 330:326, 2005 2. Adam R, Reynes M, Johann M, et al: The outcome of steatotic grafts in liver transplantation. Transplant Proc 23:1538, 1991 3. Crowley H, Lewis WD, Gordon F, et al: Steatosis in donor and transplant liver biopsies. Hum Pathol 31:1209, 2000 4. Imber CJ, St Peter SD, Handa A, et al: Hepatic steatosis and its relationship to transplantation. Liver Transpl 8:415, 2002 5. Fishbein TM, Fiel MI, Emre S, et al: Use of livers with microvesicular fat safely expands the donor pool. Transplantation 64:248, 1997 6. Nadig SN, Bratton CF, Karp SJ: Marginal donors in liver transplantation: expanding the donor pool. J Surg Educ 64:46, 2007 7. Schemmer P, Nickkholgh A, Hinz U, et al: Extended donor criteria have no negative impact on early outcome after liver transplantation: a single-center multivariate analysis. Transplant Proc 39:529, 2007 8. Nocito A, El-Badry AM, Clavien PA: When is steatosis too much for transplantation? J Hepatol 45:494, 2006 9. Garcia Urena MA, Colina Ruiz-Delgado F, Moreno Gonzalez E, et al: Hepatic steatosis in liver transplant donors: common feature of donor population? World J Surg 22:837, 1998 10. Frankel WL, Tranovich JG, Salter L, et al: The optimal number of donor biopsy sites to evaluate liver histology for transplantation. Liver Transpl 8:1044, 2002 11. Kleiner DE, Brunt EM, Van Natta M, et al: Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313, 2005
