INTRODUCTION
============

Orthotopic liver transplantation is the only choice for treatment of end-stage liver diseases. However, it is a major surgery with many complications, especially when marginal livers with prolonged ischemic time and steatosis are used \[[@B1]\]. One of the main contributing factors in primary allograft failure and poor initial function of transplanted livers is preservation injury after reperfusion of the donor organ, secondary to release of reactive oxygen species and pro-inflammatory mediators \[[@B2]\].

There have been several reported methods for the assessment of the severity and presence of preservation and reperfusion injuries, such as measuring the level of interleukins and cytokines as pro-inflammatory molecules \[[@B3]\]. One of these methods is histopathologic findings in the post-perfusion liver needle biopsies, which have rarely been investigated in the previous studies \[[@B4]\]. There have been many reported findings in favor of reperfusion injury. However, there is no well documented scoring system. Presence of hepatocellular necrosis, hepatocyte ballooning degeneration, steatosis, cholestasis, lobular neutrophilic infiltration, and hepatocellular apoptosis have been reported as main histopathologic findings in favor of reperfusion injury in post-perfusion (time zero) liver needle biopsies \[[@B2]\].

In this study, we tried to evaluate the histopathologic findings of post-perfusion, time zero liver needle biopsies in 408 transplanted livers.

PATIENTS AND METHODS
====================

From 2013 to 2015, 408 patients underwent liver transplantation with post-perfusion and time zero liver needle biopsy. Patients with hepatic artery thrombosis, biliary complications, sepsis, fungal infections, and other well-defined causes of transplant injury were excluded from the study. The patients included 268 (65.6%) males and 140 (34.3%) females. The mean±SD age of donors and recipients was 31.1±15 (range: 2--69) and 35.9±17.2 (range: 2--69) years, respectively. These biopsies have been taken within the first hour of vascular anastomosis. All of the specimens were fixed in 10% buffered formalin and embedded in paraffin, then stained by hematoxylin and eosin.

All the liver needle biopsies were examined by a hepatopathologist blinded to the outcome of the liver transplantation for the histopathologic changes of reperfusion injury, i.e., presence and the severity of steatosis, hepatocellular necrosis, neutrophilic infiltration, apoptosis, ballooning degeneration, and cholestasis as follows \[[@B5]\]: For grading of macrovesicular steatosis, we assumed grade 0 (no to \<5%), grade I (5% to \<30%), grade II (30% to \<60%), and grade III (≥60%). For hepatocellular necrosis, neutrophilic infiltration, and apoptosis, the grading system used was: absent, grade I (1 focus/HPF), grade II (2--3 foci /HPF), and grade III (\>3 foci/HPF). And for grading hepatocyte ballooning degeneration and cholestasis, we used the following grading system: absent, grade I (mild), grade II (moderate), and grade III (severe).

All the information about the donor and recipient relating to the outcome of early liver transplantation including cold and warm ischemic times (CIT and WIT), duration of post-liver transplantation hospital stay, presence of rejection, and the last liver function test before discharge were obtained from the patients' clinical charts.

Findings of the post-perfusion liver needle biopsies were compared with the above-mentioned clinical and paraclinical findings to find out the value of main histopathologic findings of post-perfusion time zero liver needle biopsy in predicting the early outcome of the liver transplants.

RESULTS
=======

We stusied 408 patients who underwent liver transplants with post perfusion, time zero liver needle biopsies. [Table 1](#T1){ref-type="table"} shows the frequency of histologic findings in these 408 post-perfusion biopsies. Different characteristics that can be indicative of early post-liver transplantation outcome were compared with the above histologic findings, i.e., the duration of hospital stay, the level of the last (before discharge) ALT, AST, alkaline phosphatase, and bilirubin level. The mean±SD AST, ALT, and alkaline phosphatase were 148.46±140.80 (range: 8--535), 141±125.37 (range: 7--399), and 147±76.37 (range: 114--236) IU/L, respectively. The mean±SD bilirubin level was 6.5±2.3 (range: 0.3--9.5) mg/dL. The mean±SD hospital stay was 13.04±8.04 (range: 10--121) days. The mean±SD cold and warm ischemic times were 462.79±170.7 (range: 15--840) and 42.85±9.04 (range: 25--80) minutes, respectively. Among the 408 studied patients, 117 patients underwent liver biopsy during their hospital stay; 68 (16.6%) of whom had various grades of acute rejection (the remaining patients were within the normal limit). Different grades of histologic characteristics were compared with different grades and levels of the above-mentioned findings such as the level of ALT and AST,*etc*.

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Frequency of histopathologic findings in 408 post-perfusion time zero biopsies

  Finding                              Grade 0       Grade I       Grade II     Grade III
  ------------------------------------ ------------- ------------- ------------ -------------
  Neutrophilic infiltration            167 (40.9%)   45 (11.0%)    46 (11.2%)   150 (36.8%)
  Steatosis                            254 (62.2%)   136 (33.3%)   17 (4.2%)    1 (0.0%)
  Hepatocellular apoptosis             304 (74.5%)   51 (12.5%)    23 (5.6%)    30 (7.4%)
  Hepatocellular necrosis              341(83.5%)    27 (6.6%)     25(6.1%)     15(3.7%)
  Hepatocyte ballooning degeneration   221(54.1%)    166 (40.7%)   17 (4.2%)    4 (1.0%)
  Cholestasis                          388 (95.1%)   10 (2.4%)     10(2.4%)     0 (0.0%)

[Table 2](#T2){ref-type="table"} shows the comparison between different grades of histologic findings and the above-mentioned characteristics.

###### 

The association between different grades of histologic findings in post-perfusion liver needle biopsies with different characteristics of liver transplant outcome in 408 studied patients

  Finding                                Grade         CIT[\*](#TFN1){ref-type="table-fn"} (minute)   WIT[†](#TFN2){ref-type="table-fn"} (minute)   Presence of Rejection   Hospital stay (day)   AST (IU/L)    ALT (IU/L)    Alk (IU/L)   Bil-Total (mg/dL)
  -------------------------------------- ------------- ---------------------------------------------- --------------------------------------------- ----------------------- --------------------- ------------- ------------- ------------ -------------------
  PMN infiltrate                         0             256±74.4                                       27±4.7                                        2                       10.6±2.3              42.8±28.6     28.6±23.3     47±33.7      0.4±2
  I                                      438.2±197.3   44.3±8.7                                       9                                             11±5.2                  69.9±38.9             65.1±29.3     37.8±42       1.4±2.5      
  II                                     374.9±138.3   41±7.6                                         6                                             20.6±6.5                158.5±118.7           133.7±120.3   112±66.3      2.5±4        
  III                                    474±166.7     42.8±9.8                                       51                                            85±7.7                  198.7±130.8           209.1±130.8   166±75.1      5.9±3.1      
  p value                                0.05          0.05                                           0.04                                          0.05                    0.05                  0.05          0.04          0.05         
  Steatosis                              0             466.1±164.5                                    42.6±9.1                                      43                      11.3±3.3              56.9±191.5    133.5±153     42.5±27.9    2.1±2.5
  I                                      454.2±183.3   43.2±9.2                                       20                                            13.5±6.1                216±131.9             218±34.9      45.4±35.1     2.7±3.5      
  II                                     459.4±169.2   42.6±7.6                                       5                                             33.4±7.2                240.6±20.2            154.7±145.8   154±145       1.6±1.3      
  III^[‡](#TFN3){ref-type="table-fn"}^   720           50                                             ---                                           14                      36                    78            236           1.3          
  p value                                0.1           0.8                                            0.1                                           0.05                    0.05                  0.1           0.05          0.1          
  Hepatocellular necrosis                0             461.6±169.4                                    42.8±9.1                                      55                      13.1±8.1              51±82.5       165±47.9      230±32       2.3±2.8
  I                                      481.7±194.1   43.4±8.4                                       4                                             12.7±6.2                48.4±82.3             102.2±73.9    114±85.9      2±2.5        
  II                                     472.2±167.8   44.3±7.6                                       6                                             14.1±5.1                110.5±81.5            123.1±234     123±42        3.2±1.9      
  III                                    432.2±186.1   42.1±6.7                                       2                                             13.4±4.1                115.3±78.9            134.2±102     114.1±76      2.3±2.9      
  p value                                0.5           0.5                                            0.6                                           0.8                     0.6                   0.4           0.3           0.5          
  Ballooning degeneration                0             458.9±169.4                                    42.5±8.5                                      39                      13.3±8.8              144.1±102.2   189.2±70.6    255±133      2.4±3.4
  I                                      468.5±178.5   43±9.1                                         27                                            12.7±7                  60.7±145.4            128.5±158.7   265±49.1      2.2±1.9      
  II                                     457.5±136.2   45±13                                          2                                             14.6±6.6                38.3±27.2             91.5±69.4     233±44.2      1.5±0.7      
  III                                    480±49        48.7±14                                        0                                             10±1.1                  29±24                 102.4±30.4    234±48.9      1.9±0.5      
  p value                                0.9           0.3                                            0.07                                          0.6                     0.8                   0.5           0.3           0.4          
  Apoptosis                              0             458.5±176.7                                    43±9.3                                        50                      11.3±3.7              127.9±69.1    177.1±130.2   48.7±36.1    2.3±2.9
  I                                      479.3±140.9   41.9±8.4                                       12                                            14.7±8                  35.3±17.2             97.5±84.4     34.4±30.9     2.7±3.2      
  II                                     466.4±146.3   43.2±7.6                                       1                                             16.1±16                 41±33.9               98.2±80.7     39.2±165      1.9±1.7      
  III                                    478.6±173.2   42.1±7.9                                       5                                             12.6±6.1                55.7±78.2             42.8±99.8     45.7±133      2.1±2.9      
  p value                                0.8           0.8                                            0.06                                          0.013                   0.8                   0.6           0.8           0.6          
  Cholestasis                            0             466.4±168.8                                    42.9±9.1                                      62                      13±8.1                110±86.9      163.4±48.4    236.1±49     2.3±2.8
  I                                      419.3±189.2   43.1±8.1                                       6                                             13.7±7.5                71.2±140.1            148.9±32.1    250.9±34      2.5±3.4      
  II                                     450±196.2     36.7±7.5                                       0                                             9.5±1.6                 66.5±95.8             73±47.6       301±24.5      2.3±3.9      
  III[§](#TFN4){ref-type="table-fn"}     ---           ---                                            ---                                           ---                     ---                   ---           ---           ---          
  p value                                0.3           0.2                                            0.4                                           0.4                     0.9                   0.9           0.8           0.8          

CIT: Cold ischemic time

WIT: Warm ischemic time

There was only one patient transplanted with more than 60% steatosis in the post-perfusion liver needle biopsy

There was no patient with severe cholestasis (grade III) in the biopsies examined

DISCUSSION
==========

Liver transplantation is the main treatment for patients with end-stage liver failure. In spite of the high success rate and survival rate in this procedure, there are still early complications. One of these complications is attributed to reperfusion injury \[[@B6]\]. There are very few studies regarding histopathologic features of reperfusion injury, predicting by post-reperfusion, time zero liver needle biopsy, which is taken within one hour after vascular anastomosis in liver transplantation \[[@B6], [@B7]\]. One of the strengths of the current study is its sample size: all previous studies have been performed on less than 200 cases; in the currents study, we included 408 patients.

We observed significant (p\<0.05) correlation between the presence of neutrophils in the liver with CIT, WIT, rejection, duration of hospital stay and also early LFT levels. This finding was also shown in some of the previous studies as the most important characteristic predicting reperfusion injury \[[@B2]\]. In some reports, presence of hepatocellular necrosis has been introduced as one of the main features of severe reperfusion injury and correlated with early post-liver transplant outcome \[[@B8]\]. However, in our study, the majority of cases (83.5%) failed to show any evidence of hepatocellular necrosis and the correlation between this histopathologic finding and other features was not significant. This was also true for liver steatosis, i.e., more than 80% of our patients showed less than 30% of steatosis with statistically significant correlation with post-liver transplantation AST and alkaline phosphatase level as well as duration of hospital stay (p\<0.05). In some reports, steatosis did not show any major effect on the outcome of liver transplantation after seven days; the authors believe that the presence of steatosis in post-reperfusion liver needle biopsy has minor effects on the LFT and other clinical findings in the first seven days \[[@B9], [@B10]\]. In the studied 408 patients, about 25% of the biopsies showed different grades of hepatocellular apoptosis. However, there was no correlation between these findings and early post-liver transplantation complications. In some of the previous studies, this finding was well correlated with post-liver transplant LFT levels \[[@B1]\]. We also evaluated hepatocyte ballooning degeneration and cholestasis, none of which was correlated with early post-liver transplant findings in favor of reperfusion injury.

In conclusion, the most important and predicting histopathologic finding in post-perfusion liver needle biopsy was infiltration of neutrophils, which should be highly appreciated in pathology report and should be considered an important high-risk sign by the clinicians.

This project was registered as project No. 5090 in Shiraz University of Medical Sciences, Shiraz, Iran.

FINANCIAL SUPPORT:
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None.
