ORIGINAL ARTICLE: HEPATOLOGY
Fulminant Hepatic Failure of Autoimmune Aetiology in Children
A. Di Giorgio, M. Bravi, yE. Bonanomi, zG. Alessio, §A. Sonzogni, jjY. Zen, ô M. Colledan, and L. D’Antiga
ABSTRACT Objective: Autoimmune hepatitis (AIH) is considered an underdiagnosed cause of fulminant hepatic failure (FHF). Autoimmune FHF (AI-FHF) is believed to lead invariably to liver transplantation (LTX) or death. We aimed to describe the autoimmune features of children diagnosed as having AI-FHF and indeterminate FHF (ID-FHF), and describe the outcome of patients with AI-FHF treated with immunosuppressive drugs. Methods: In this case–control study, the files of patients with AI-FHF and ID-FHF were reviewed and compared. AIH was diagnosed based on positive autoantibodies, raised immunoglobulin G, and histology when available. FHF was defined by raised transaminases, international normalised ratio 2.0, presence of encephalopathy, and no previously recognised liver disease. Results: A total of 46 children with FHF were managed in the last 15 years: 10/46 (22%) had AI-FHF, 20/46 (43%) ID-FHF, and 16 had other diagnosis. The mean follow-up time was 4.6 years. AI-FHF and ID-FHF differed for the presence of autoantibodies (10/10, 6/10 liver/kidney microsome [LKM]type, vs 3/20, none LKM, P < 0.0001), immunoglobulin G level (1845 vs 880 mg/dL, P < 0.001), median age at diagnosis (6.4 vs 1.8 years, P ¼ 0.017), and alanine aminotransferase level (1020 vs 2386 IU/L, P ¼ 0.029). Liver histology did not allow to differentiate the 2 conditions. Among the patients with AI-FHF, 4/9 who received steroids recovered; 5/9 required LTX and 1 died awaiting treatment. Conclusions: AIH is a much more common cause of FHF than previously suggested, and a complete autoantibody testing including LKM-type is essential in this setting. Autoantibodies are uncommon in ID-FHF, and histology cannot distinguish it from AI-FHF. A cautious steroid trial may avoid LTX in some of the patients with AI-FHF. Key Words: autoantibodies, autoimmune hepatitis, children, fulminant hepatic failure, indeterminate acute liver failure, liver transplantation
autoantibodies, elevated serum immunoglobulin G (IgG), and histological features of interface hepatitis (1). Up to 30% to 40% of children with AIH can have an acute presentation, but a minority of them have a fulminant course characterised by encephalopathy and need for liver transplantation (LTX) (2–4). The cause of fulminant hepatic failure (FHF) in children can be determined in approximately half of patients, whereas in the other half the condition remains indeterminate (5). In particular, the autoimmune aetiology may be underestimated. AIH represents a reversible cause of FHF, and in adults the early identification and treatment with steroids have been shown to avoid LTX in approximately one-third of patients (6–8). Nonetheless in patients with suspected yet unproven autoimmune FHF (AI-FHF), a blind trial of steroids exposes the patient to an increased risk of sepsis (9). FHF represents a life-threatening event in which coagulopathy often contraindicates liver biopsy, the mainstay of the diagnosis of AIH. The picture is made further complicated by the fact that autoantibodies may be negative at presentation, and antinuclear autoantibodies (ANAs) as well as smooth muscle autoantibodies (SMAs) can be positive even in patients with liver diseases of different aetiologies (10–12). We aimed to describe the clinical features of children with AI-FHF, compare them with those labelled as indeterminate FHF (ID-FHF), and observe whether immunosuppressive treatment may affect the course of the disease and avoid LTX.
METHODS Patient Selection and Clinical Data
(JPGN 2015;60: 159–164)
A
utoimmune hepatitis (AIH) is a chronic and progressive liver disease affecting all age groups, with a higher incidence among girls. The hallmarks of AIH are the presence of
Received June 27, 2014; accepted October 3, 2014. From Paediatric Hepatology, Gastroenterology and Transplantation, the yPaediatric Intensive Care Unit, the zLaboratory Medicine, §Transplant Pathology, Hospital Papa Giovanni XXIII, Bergamo, Italy, the jjPathology Department, Institute of Liver Studies King’s College Hospital, London, UK, and ôGeneral Surgery and Transplantation, Hospital Papa Giovanni XXIII, Bergamo, Italy. Address correspondence and reprint requests to Lorenzo D’Antiga, MD, Paediatric Hepatology, Gastroenterology and Transplantation, Hospital Ospedaliera Papa Giovanni XXIII, Piazza Oms 1, 24127 Bergamo, Italy (e-mail: [email protected]). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000593
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Volume 60, Number 2, February 2015
We reviewed retrospectively all of the FHF cases referred to our centre between 1996 and 2012, excluding those with a neonatal onset. We collected demographic data, presence of hepatic encephalopathy (HE), peak of serum levels of alanine aminotransferase (ALT), total bilirubin, ammonia, prothrombin time, international normalised ratio (INR), IgG, immunoglobulin M (IgM), presence of autoantibodies (anti-SMA, ANA, anti–liver/kidney microsomes [LKM]-1, anti-liver cytosol, LC1), histological features, immunosuppressive therapy, LTX, outcome. We also tested IgG/IgM ratio, because in AI-FHF IgG is expected to be raised, whereas in ID-FHF (hypothetically as a result of an unidentified infection) IgM may be raised, increasing the power of this ratio in distinguishing the 2 conditions (13). Conventionally, the diagnosis of acute liver failure (ALF) in children is based on the biochemical evidence of acute liver injury, coagulopathy (INR 2.0), and no previously recognised chronic liver disease regardless of encephalopathy (HE) (5). In this study, we included only those patients presenting with the most severe end of the spectrum of ALF, characterised also by the presence of HE (FHF). HE has been evaluated on physical examination and using electroencephalogram (14,15).
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Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
D’Antiga et al To identify the aetiology of FHF, the patients underwent a wide diagnostic workup, including viral, toxic, and metabolic conditions. History of drug-induced (DI) hepatotoxicity or mushroom poisoning was searched for in all of the patients. FHF owing to Wilson disease was excluded by family history and age (8 years of age. When the tests could not identify a cause, the patients were considered affected by ID-FHF. The original diagnosis of AI-FHF had been made at presentation, and was based on the classical criteria used to diagnose AIH in children (high transaminases and IgG, presence of autoantibodies, compatible histology when available) (1). AIH was classified according to seropositivity for SMA and/or ANA antibody (AIH type 1, AIH-1), or LKM-1 and/or LC1 (AIH type 2, AIH-2). As reported in adults, we also retrospectively applied to the 2 cohorts of patients the simplified diagnostic criteria (SDC) developed by the International Autoimmune Hepatitis Group (IAIHG), and considered as the criterion standard for the diagnosis of AIH but not standardised in AI-FHF (16,17). This system includes the following: 1. Autoantibody positivity (up to 2 points) 2. IgG levels above the upper limit of normal (up to 2 points; this was corrected for the paediatric normal values) (18) 3. Compatible histology (up to 2 points) 4. Exclusion of other diagnosis (2 points)
Histological Evaluation The histology samples were reviewed blindly and independently by 2 expert liver pathologists (A.S., Y.Z., working in different centres and unaware of the clinical features of the patients studied) and scored according to 2 systems: the classical assessment developed by the IAIHG and more recently revised (16,17), and the system pointing at centrilobular disease and different patterns of massive hepatic necrosis (MHN) designed specifically for AI-FHF (11,12,19). In this system MHN1 represents the classical massive necrosis with near-complete loss of hepatocytes throughout the lobules, residual intrasinusoidal inflammation, periportal neocholangiolar proliferation (ductular reaction), and portal/periportal inflammation. MHN2 includes areas of submassive necrosis, representing regions of MHN1 as well as regenerative nodules and areas of early fibrosis; it is seen in more subacute clinical courses than MHN1. MHN3 demonstrates necroinflammatory changes of acute hepatitis in portions of the specimen (spotty necrosis) as well as other regions with more substantial confluent necrosis, including areas of bridging hepatic necrosis or multilobular necrosis with neocholangiolar proliferation. MHN4 shows a panlobular necrosis, but with prominence of centrilobular necroinflammation and haemorrhage, resembling the severe form of the centrilobular variant of AIH and the centrilobular variant of acute cellular rejection observed in transplant allografts. MHN5 represents the classical periportal AIH in conjunction with superimposed changes of massive necrosis and sometimes centrilobular necroinflammation. According to this classification the patterns MHN1, MHN2, and MHN3 are considered relatively nonspecific, whereas MHN4 and MHN5 are considered more characteristic of an autoimmune pathogenesis (11).
Autoantibody Detection Autoantibody testing (ANA, SMA, LKM-1, anti–mitochondrial antibodies [AMA], and LC1) was carried out by indirect
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immunofluorescence (IFL) on cryostat sections of mouse liver and kidney and stomach specimens (Inova, San Diego, CA) at serial dilutions (1:20, 1:40, 1:80, 1:160) to avoid a prozone effect (High Dose Hook effect) caused by an excess amount of antibody. To further characterise the pattern associated with ANA, all ANA samples were retested by IFL on human laryngeal carcinoma (HEp-2) cells, serum dilutions from 1:20 to 1:320 (HEp-2; Inova). ANA-positive serum samples were also tested for quantitative anti– double-stranded DNA (Thermo Fisher Scientific, Waltham, MA), IFL assay by using the Crithidia luciliae (Inova) and the anti– extractable nuclear antigen (anti-ENA) reactivity (Thermo Fisher Scientific). IFL was performed by 2 expert investigators who were blinded to each other’s findings and to each patient’s clinical diagnosis. Immunoblot assay (Euroassay Liver Profile, EUROIMMUN, Lu¨beck, Germany) was also performed on each sample as a confirmatory test for the presence of LKM-1 or AMA in IFL and to assess the possible coexistence of soluble liver antigen (SLA), and LC1 positivity in LKM-1–positive patients.
Statistical Analysis Data are reported as medians and ranges unless specified differently. The Student t test for paired samples and the Fisher exact test for differences between groups were used. The analysis was performed with SPSS version 13.0 (SPSS Inc, Chicago, IL) for Windows. A P 0.05 or less was assigned significance.
RESULTS In our database we found 46 children diagnosed as having FHF (M/F 26/20, median age at presentation 3.1 years, range 0.2– 15.1 years). In the same period 440 patients underwent LTX at our centre. Ten of 46 (22%) (M/F 6/4) patients with FHF had been diagnosed as having AI-FHF (group 1, G1), 20 (43%) as having ID-FHF (group 2, G2), whereas 16/46 (35%) had a different diagnosis, including acetaminophen overdose (n ¼ 6), metabolic disorders (n ¼ 3), Wilson disease (n ¼ 3), mushroom poisoning (n ¼ 3), and viral infection (n ¼ 1). All of the patients were tested for autoantibodies. The mean follow-up time was 4.6 years. In G1, urinary copper excretion was >100 mg/day in only 1 patient of 3 years of age. In 3 patients >8 years of age, Wilson disease was excluded by normal ceruloplasmin and urinary copper 50,000/mL and with any value of INR, following a transfusion of fresh frozen plasma. There were no major complications after the procedure in these patients. At retrospective revision of the patients according to the SDC criteria, 8/10 fulfilled the criteria, whereas 2 did not, with a borderline score of 5 (patients 3 and 8, Table 3). Patient 3 did not receive steroids, was transplanted, and autoimmune disease could not be confirmed during the follow-up, whereas patient 8 responded rapidly to steroids, was not transplanted, and the disease was confirmed at follow-up biopsies. Nine of 10 patients received immunosuppressive treatment according to the recommendations previously reported for AIH (1): methylprednisolone 2 mg kg1 day1 (up to daily dose of 60 mg) IV for 14 days and then tapered down to a maintenance dose of 5 or 2.5 mg/day (depending on the patient weight) during a 4-week www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 2, February 2015
Autoimmune Fulminant Hepatic Failure in Children
TABLE 1. Comparison of clinical features, laboratory investigations at presentation, and outcome in children with FHF of autoimmune and indeterminate aetiology Group 1 (n ¼ 10) autoimmune
Clinical features/laboratory investigations Age at onset ALT, IU/L Total bilirubin, mg/dL INR Ammonia, mmol/L IgG, mg/dL IgM, mg/dL IgG/ULN IgG/IgM ratio Female prevalence, % Prevalence of autoantibodies, % LTX, % Overall survival, %
Group 2 (n ¼ 20) indeterminate
7.6 (4.8) 1140 (459) 19 (10.8) 3.6 (0.8) 191 (85) 2070 (114) 166 (91) 1.6 (0.8) 13.7 (6.6) 40 100 50 90
3.5 3086 23.0 4.7 179 948 126 1.01 8.2
P
(3.9) (2637) (2.0–46.1) (1.66) (84) (388) (52) (0.58) (4.2) 55 15 70 90
0.017 0.029 0.637 0.130 0.716 2000 IU/L in 1/ 10 patients of group 1 versus 14/20 patients of group 2 (P ¼ 0.005, 2-tailed Fisher exact test).
Histological Evaluation Histology was available in 83% of patients. The agreement between the 2 pathologists in diagnosing autoimmune features was 75%. When the evaluation was not in full agreement, we used the highest score proposed by the 2 pathologists for each patient (Table 3). All of the 9 specimens from patients in G1 resulted compatible or typical of AIH (score 1 or 2 according to the SDC of the IAIHG); among G2 patients, 12/16 specimens were compatible or typical of AIH, making a positive predictive value (PPV) of 42% and a negative predictive value (NPV) of 100% for this evaluation. Looking exclusively at the typical histology (score 2 in SDC classification), the PPV became 83% and the NPV 79% (5/9 G1 patients and 1/16 G2 patients), suggesting that in this setting histology is helpful when strongly consistent with AIH. For the MHN classification 6/9 patients in G1 had a pattern suggestive of AI-FHF (MHN4 or MHN5), versus 8/16 in G2 (PPV 43%, NPV 73%), suggesting that this classification was poorly reliable in our study group.
DISCUSSION Identifying AIH as the aetiology of FHF is an unsolved clinical issue (21). Because of the rarity of the disease the diagnosis of AI-FHF is a challenge in both adults and children, and it is likely that the condition is overlooked. In previous reports viral infections and autoimmune diseases were underestimated in this setting so that many patients ended up with a diagnosis of ID-FHF (11,20,22–27). This may be because in some centres autoantibodies are not routinely measured in FHF. In this study, in which all of the patients were tested for autoantibodies at presentation, there was minimal overlap between AI-FHF and indeterminate cases. In our study we found a higher percentage of patients with an autoimmune cause of FHF as compared with previous studies (2,28,29). This may be because of our high level of suspicion or be peculiar to the Italian population. Alternatively, although our series is the largest reported so far, this could be related to the small sample size. The higher prevalence of AIH-2 presenting as FHF in children confirms what has been previously reported (30). Because our centre is a referral unit for LTX, our patients represent the most severe end of the spectrum of AI-FHF. Many AIH with an acute presentation would fit the criteria set by the pediatric acute liver failure study group (5), but only a few present www.jpgn.org
Autoimmune Fulminant Hepatic Failure in Children with encephalopathy and a life-threatening condition; in these patients making a timely diagnosis is much more difficult. In this retrospective study, the strongest feature of autoimmune liver disease remains the presence of autoantibodies (especially anti-LKM that are distinctive of AIH) and raised IgG. In a large cohort of children reported by Gregorio et al (30), it was shown that only 2/32 patients with AIH-1 and 1/20 with AIH-2 were seronegative at presentation and became positive shortly after the treatment start. This suggests that autoantibody serology is a reliable marker to diagnose AIH even at the clinical onset of the disease. It is well known that a proportion of children with AIH have normal IgG levels and some with FHF of nonautoimmune aetiology can have raised IgG (31,32). Nevertheless, in our cohort IgG level remained strikingly higher in children diagnosed as having AI-FHF, even after correction by age. Because in children it is impractical to adjust IgG level for age—there is a wide variability of such tests in normal subjects during childhood—we also tested the IgG/IgM ratio; in doing so the value is normalised for the subject and the discrimination power of this test is enhanced because in ID-FHF (hypothetically as a result of an unidentified infection) IgM may be raised (13). Indeed, Bernal et al (24) showed that IgM levels are higher in adult patients with FHF because of a viral infection compared with those with FHF from other causes. We found that the IgG/IgM ratio was more commonly abnormal than IgG corrected for age in our cohort of patients with AI-FHF, suggesting that this test could be adopted in the place of IgG. Liver biopsy is the mainstay to make the diagnosis of AIH but, in the setting of FHF, it is often unfeasible because of the risk of bleeding. In our centre, however, liver biopsy has been performed even in patients with ALF, providing important information on histological features of FHF. It is well known that in FHF the classical histological pattern of AIH can be masked, mixed, or replaced by a picture of massive necrosis and/or multilobular collapse, which is a common feature in other causes of FHF (5,6,10,13,33,34). Stravitz et al (11) studied a group of 72 adult patients with ID-FHF and proposed a histological classification that pointed at centrilobular disease (MHN4 and MHN5) as a feature of AI-FHF. In our study we could not demonstrate a greater prevalence of MHN4–5 in patients with AI-FHF compared with those with ID-FHF. Histological evaluation was more helpful when scored according to SDC criteria, although we were unable to show a clearcut difference between G1 and G2 patients, both for the SDC and for the MHN histology classifications. Histology can differentiate well AI-FHF when the features are typical or absent according to the SDC criteria, whereas it has low diagnostic power if the pattern is less striking, as occurred in many of our patients, questioning the utility of performing a dangerous liver biopsy in the presence of severe coagulopathy and encephalopathy. AIH tends to develop and present later in childhood than other paediatric liver disorders, which often have a genetic cause (35,36). Several authors have regarded AIH as a rare cause of FHF in children
Fulminant Hepatic Failure of Autoimmune Aetiology in Children
A. Di Giorgio, M. Bravi, yE. Bonanomi, zG. Alessio, §A. Sonzogni, jjY. Zen, ô M. Colledan, and L. D’Antiga
ABSTRACT Objective: Autoimmune hepatitis (AIH) is considered an underdiagnosed cause of fulminant hepatic failure (FHF). Autoimmune FHF (AI-FHF) is believed to lead invariably to liver transplantation (LTX) or death. We aimed to describe the autoimmune features of children diagnosed as having AI-FHF and indeterminate FHF (ID-FHF), and describe the outcome of patients with AI-FHF treated with immunosuppressive drugs. Methods: In this case–control study, the files of patients with AI-FHF and ID-FHF were reviewed and compared. AIH was diagnosed based on positive autoantibodies, raised immunoglobulin G, and histology when available. FHF was defined by raised transaminases, international normalised ratio 2.0, presence of encephalopathy, and no previously recognised liver disease. Results: A total of 46 children with FHF were managed in the last 15 years: 10/46 (22%) had AI-FHF, 20/46 (43%) ID-FHF, and 16 had other diagnosis. The mean follow-up time was 4.6 years. AI-FHF and ID-FHF differed for the presence of autoantibodies (10/10, 6/10 liver/kidney microsome [LKM]type, vs 3/20, none LKM, P < 0.0001), immunoglobulin G level (1845 vs 880 mg/dL, P < 0.001), median age at diagnosis (6.4 vs 1.8 years, P ¼ 0.017), and alanine aminotransferase level (1020 vs 2386 IU/L, P ¼ 0.029). Liver histology did not allow to differentiate the 2 conditions. Among the patients with AI-FHF, 4/9 who received steroids recovered; 5/9 required LTX and 1 died awaiting treatment. Conclusions: AIH is a much more common cause of FHF than previously suggested, and a complete autoantibody testing including LKM-type is essential in this setting. Autoantibodies are uncommon in ID-FHF, and histology cannot distinguish it from AI-FHF. A cautious steroid trial may avoid LTX in some of the patients with AI-FHF. Key Words: autoantibodies, autoimmune hepatitis, children, fulminant hepatic failure, indeterminate acute liver failure, liver transplantation
autoantibodies, elevated serum immunoglobulin G (IgG), and histological features of interface hepatitis (1). Up to 30% to 40% of children with AIH can have an acute presentation, but a minority of them have a fulminant course characterised by encephalopathy and need for liver transplantation (LTX) (2–4). The cause of fulminant hepatic failure (FHF) in children can be determined in approximately half of patients, whereas in the other half the condition remains indeterminate (5). In particular, the autoimmune aetiology may be underestimated. AIH represents a reversible cause of FHF, and in adults the early identification and treatment with steroids have been shown to avoid LTX in approximately one-third of patients (6–8). Nonetheless in patients with suspected yet unproven autoimmune FHF (AI-FHF), a blind trial of steroids exposes the patient to an increased risk of sepsis (9). FHF represents a life-threatening event in which coagulopathy often contraindicates liver biopsy, the mainstay of the diagnosis of AIH. The picture is made further complicated by the fact that autoantibodies may be negative at presentation, and antinuclear autoantibodies (ANAs) as well as smooth muscle autoantibodies (SMAs) can be positive even in patients with liver diseases of different aetiologies (10–12). We aimed to describe the clinical features of children with AI-FHF, compare them with those labelled as indeterminate FHF (ID-FHF), and observe whether immunosuppressive treatment may affect the course of the disease and avoid LTX.
METHODS Patient Selection and Clinical Data
(JPGN 2015;60: 159–164)
A
utoimmune hepatitis (AIH) is a chronic and progressive liver disease affecting all age groups, with a higher incidence among girls. The hallmarks of AIH are the presence of
Received June 27, 2014; accepted October 3, 2014. From Paediatric Hepatology, Gastroenterology and Transplantation, the yPaediatric Intensive Care Unit, the zLaboratory Medicine, §Transplant Pathology, Hospital Papa Giovanni XXIII, Bergamo, Italy, the jjPathology Department, Institute of Liver Studies King’s College Hospital, London, UK, and ôGeneral Surgery and Transplantation, Hospital Papa Giovanni XXIII, Bergamo, Italy. Address correspondence and reprint requests to Lorenzo D’Antiga, MD, Paediatric Hepatology, Gastroenterology and Transplantation, Hospital Ospedaliera Papa Giovanni XXIII, Piazza Oms 1, 24127 Bergamo, Italy (e-mail: [email protected]). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000593
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Volume 60, Number 2, February 2015
We reviewed retrospectively all of the FHF cases referred to our centre between 1996 and 2012, excluding those with a neonatal onset. We collected demographic data, presence of hepatic encephalopathy (HE), peak of serum levels of alanine aminotransferase (ALT), total bilirubin, ammonia, prothrombin time, international normalised ratio (INR), IgG, immunoglobulin M (IgM), presence of autoantibodies (anti-SMA, ANA, anti–liver/kidney microsomes [LKM]-1, anti-liver cytosol, LC1), histological features, immunosuppressive therapy, LTX, outcome. We also tested IgG/IgM ratio, because in AI-FHF IgG is expected to be raised, whereas in ID-FHF (hypothetically as a result of an unidentified infection) IgM may be raised, increasing the power of this ratio in distinguishing the 2 conditions (13). Conventionally, the diagnosis of acute liver failure (ALF) in children is based on the biochemical evidence of acute liver injury, coagulopathy (INR 2.0), and no previously recognised chronic liver disease regardless of encephalopathy (HE) (5). In this study, we included only those patients presenting with the most severe end of the spectrum of ALF, characterised also by the presence of HE (FHF). HE has been evaluated on physical examination and using electroencephalogram (14,15).
159
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
D’Antiga et al To identify the aetiology of FHF, the patients underwent a wide diagnostic workup, including viral, toxic, and metabolic conditions. History of drug-induced (DI) hepatotoxicity or mushroom poisoning was searched for in all of the patients. FHF owing to Wilson disease was excluded by family history and age (8 years of age. When the tests could not identify a cause, the patients were considered affected by ID-FHF. The original diagnosis of AI-FHF had been made at presentation, and was based on the classical criteria used to diagnose AIH in children (high transaminases and IgG, presence of autoantibodies, compatible histology when available) (1). AIH was classified according to seropositivity for SMA and/or ANA antibody (AIH type 1, AIH-1), or LKM-1 and/or LC1 (AIH type 2, AIH-2). As reported in adults, we also retrospectively applied to the 2 cohorts of patients the simplified diagnostic criteria (SDC) developed by the International Autoimmune Hepatitis Group (IAIHG), and considered as the criterion standard for the diagnosis of AIH but not standardised in AI-FHF (16,17). This system includes the following: 1. Autoantibody positivity (up to 2 points) 2. IgG levels above the upper limit of normal (up to 2 points; this was corrected for the paediatric normal values) (18) 3. Compatible histology (up to 2 points) 4. Exclusion of other diagnosis (2 points)
Histological Evaluation The histology samples were reviewed blindly and independently by 2 expert liver pathologists (A.S., Y.Z., working in different centres and unaware of the clinical features of the patients studied) and scored according to 2 systems: the classical assessment developed by the IAIHG and more recently revised (16,17), and the system pointing at centrilobular disease and different patterns of massive hepatic necrosis (MHN) designed specifically for AI-FHF (11,12,19). In this system MHN1 represents the classical massive necrosis with near-complete loss of hepatocytes throughout the lobules, residual intrasinusoidal inflammation, periportal neocholangiolar proliferation (ductular reaction), and portal/periportal inflammation. MHN2 includes areas of submassive necrosis, representing regions of MHN1 as well as regenerative nodules and areas of early fibrosis; it is seen in more subacute clinical courses than MHN1. MHN3 demonstrates necroinflammatory changes of acute hepatitis in portions of the specimen (spotty necrosis) as well as other regions with more substantial confluent necrosis, including areas of bridging hepatic necrosis or multilobular necrosis with neocholangiolar proliferation. MHN4 shows a panlobular necrosis, but with prominence of centrilobular necroinflammation and haemorrhage, resembling the severe form of the centrilobular variant of AIH and the centrilobular variant of acute cellular rejection observed in transplant allografts. MHN5 represents the classical periportal AIH in conjunction with superimposed changes of massive necrosis and sometimes centrilobular necroinflammation. According to this classification the patterns MHN1, MHN2, and MHN3 are considered relatively nonspecific, whereas MHN4 and MHN5 are considered more characteristic of an autoimmune pathogenesis (11).
Autoantibody Detection Autoantibody testing (ANA, SMA, LKM-1, anti–mitochondrial antibodies [AMA], and LC1) was carried out by indirect
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immunofluorescence (IFL) on cryostat sections of mouse liver and kidney and stomach specimens (Inova, San Diego, CA) at serial dilutions (1:20, 1:40, 1:80, 1:160) to avoid a prozone effect (High Dose Hook effect) caused by an excess amount of antibody. To further characterise the pattern associated with ANA, all ANA samples were retested by IFL on human laryngeal carcinoma (HEp-2) cells, serum dilutions from 1:20 to 1:320 (HEp-2; Inova). ANA-positive serum samples were also tested for quantitative anti– double-stranded DNA (Thermo Fisher Scientific, Waltham, MA), IFL assay by using the Crithidia luciliae (Inova) and the anti– extractable nuclear antigen (anti-ENA) reactivity (Thermo Fisher Scientific). IFL was performed by 2 expert investigators who were blinded to each other’s findings and to each patient’s clinical diagnosis. Immunoblot assay (Euroassay Liver Profile, EUROIMMUN, Lu¨beck, Germany) was also performed on each sample as a confirmatory test for the presence of LKM-1 or AMA in IFL and to assess the possible coexistence of soluble liver antigen (SLA), and LC1 positivity in LKM-1–positive patients.
Statistical Analysis Data are reported as medians and ranges unless specified differently. The Student t test for paired samples and the Fisher exact test for differences between groups were used. The analysis was performed with SPSS version 13.0 (SPSS Inc, Chicago, IL) for Windows. A P 0.05 or less was assigned significance.
RESULTS In our database we found 46 children diagnosed as having FHF (M/F 26/20, median age at presentation 3.1 years, range 0.2– 15.1 years). In the same period 440 patients underwent LTX at our centre. Ten of 46 (22%) (M/F 6/4) patients with FHF had been diagnosed as having AI-FHF (group 1, G1), 20 (43%) as having ID-FHF (group 2, G2), whereas 16/46 (35%) had a different diagnosis, including acetaminophen overdose (n ¼ 6), metabolic disorders (n ¼ 3), Wilson disease (n ¼ 3), mushroom poisoning (n ¼ 3), and viral infection (n ¼ 1). All of the patients were tested for autoantibodies. The mean follow-up time was 4.6 years. In G1, urinary copper excretion was >100 mg/day in only 1 patient of 3 years of age. In 3 patients >8 years of age, Wilson disease was excluded by normal ceruloplasmin and urinary copper 50,000/mL and with any value of INR, following a transfusion of fresh frozen plasma. There were no major complications after the procedure in these patients. At retrospective revision of the patients according to the SDC criteria, 8/10 fulfilled the criteria, whereas 2 did not, with a borderline score of 5 (patients 3 and 8, Table 3). Patient 3 did not receive steroids, was transplanted, and autoimmune disease could not be confirmed during the follow-up, whereas patient 8 responded rapidly to steroids, was not transplanted, and the disease was confirmed at follow-up biopsies. Nine of 10 patients received immunosuppressive treatment according to the recommendations previously reported for AIH (1): methylprednisolone 2 mg kg1 day1 (up to daily dose of 60 mg) IV for 14 days and then tapered down to a maintenance dose of 5 or 2.5 mg/day (depending on the patient weight) during a 4-week www.jpgn.org
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
JPGN
Volume 60, Number 2, February 2015
Autoimmune Fulminant Hepatic Failure in Children
TABLE 1. Comparison of clinical features, laboratory investigations at presentation, and outcome in children with FHF of autoimmune and indeterminate aetiology Group 1 (n ¼ 10) autoimmune
Clinical features/laboratory investigations Age at onset ALT, IU/L Total bilirubin, mg/dL INR Ammonia, mmol/L IgG, mg/dL IgM, mg/dL IgG/ULN IgG/IgM ratio Female prevalence, % Prevalence of autoantibodies, % LTX, % Overall survival, %
Group 2 (n ¼ 20) indeterminate
7.6 (4.8) 1140 (459) 19 (10.8) 3.6 (0.8) 191 (85) 2070 (114) 166 (91) 1.6 (0.8) 13.7 (6.6) 40 100 50 90
3.5 3086 23.0 4.7 179 948 126 1.01 8.2
P
(3.9) (2637) (2.0–46.1) (1.66) (84) (388) (52) (0.58) (4.2) 55 15 70 90
0.017 0.029 0.637 0.130 0.716 2000 IU/L in 1/ 10 patients of group 1 versus 14/20 patients of group 2 (P ¼ 0.005, 2-tailed Fisher exact test).
Histological Evaluation Histology was available in 83% of patients. The agreement between the 2 pathologists in diagnosing autoimmune features was 75%. When the evaluation was not in full agreement, we used the highest score proposed by the 2 pathologists for each patient (Table 3). All of the 9 specimens from patients in G1 resulted compatible or typical of AIH (score 1 or 2 according to the SDC of the IAIHG); among G2 patients, 12/16 specimens were compatible or typical of AIH, making a positive predictive value (PPV) of 42% and a negative predictive value (NPV) of 100% for this evaluation. Looking exclusively at the typical histology (score 2 in SDC classification), the PPV became 83% and the NPV 79% (5/9 G1 patients and 1/16 G2 patients), suggesting that in this setting histology is helpful when strongly consistent with AIH. For the MHN classification 6/9 patients in G1 had a pattern suggestive of AI-FHF (MHN4 or MHN5), versus 8/16 in G2 (PPV 43%, NPV 73%), suggesting that this classification was poorly reliable in our study group.
DISCUSSION Identifying AIH as the aetiology of FHF is an unsolved clinical issue (21). Because of the rarity of the disease the diagnosis of AI-FHF is a challenge in both adults and children, and it is likely that the condition is overlooked. In previous reports viral infections and autoimmune diseases were underestimated in this setting so that many patients ended up with a diagnosis of ID-FHF (11,20,22–27). This may be because in some centres autoantibodies are not routinely measured in FHF. In this study, in which all of the patients were tested for autoantibodies at presentation, there was minimal overlap between AI-FHF and indeterminate cases. In our study we found a higher percentage of patients with an autoimmune cause of FHF as compared with previous studies (2,28,29). This may be because of our high level of suspicion or be peculiar to the Italian population. Alternatively, although our series is the largest reported so far, this could be related to the small sample size. The higher prevalence of AIH-2 presenting as FHF in children confirms what has been previously reported (30). Because our centre is a referral unit for LTX, our patients represent the most severe end of the spectrum of AI-FHF. Many AIH with an acute presentation would fit the criteria set by the pediatric acute liver failure study group (5), but only a few present www.jpgn.org
Autoimmune Fulminant Hepatic Failure in Children with encephalopathy and a life-threatening condition; in these patients making a timely diagnosis is much more difficult. In this retrospective study, the strongest feature of autoimmune liver disease remains the presence of autoantibodies (especially anti-LKM that are distinctive of AIH) and raised IgG. In a large cohort of children reported by Gregorio et al (30), it was shown that only 2/32 patients with AIH-1 and 1/20 with AIH-2 were seronegative at presentation and became positive shortly after the treatment start. This suggests that autoantibody serology is a reliable marker to diagnose AIH even at the clinical onset of the disease. It is well known that a proportion of children with AIH have normal IgG levels and some with FHF of nonautoimmune aetiology can have raised IgG (31,32). Nevertheless, in our cohort IgG level remained strikingly higher in children diagnosed as having AI-FHF, even after correction by age. Because in children it is impractical to adjust IgG level for age—there is a wide variability of such tests in normal subjects during childhood—we also tested the IgG/IgM ratio; in doing so the value is normalised for the subject and the discrimination power of this test is enhanced because in ID-FHF (hypothetically as a result of an unidentified infection) IgM may be raised (13). Indeed, Bernal et al (24) showed that IgM levels are higher in adult patients with FHF because of a viral infection compared with those with FHF from other causes. We found that the IgG/IgM ratio was more commonly abnormal than IgG corrected for age in our cohort of patients with AI-FHF, suggesting that this test could be adopted in the place of IgG. Liver biopsy is the mainstay to make the diagnosis of AIH but, in the setting of FHF, it is often unfeasible because of the risk of bleeding. In our centre, however, liver biopsy has been performed even in patients with ALF, providing important information on histological features of FHF. It is well known that in FHF the classical histological pattern of AIH can be masked, mixed, or replaced by a picture of massive necrosis and/or multilobular collapse, which is a common feature in other causes of FHF (5,6,10,13,33,34). Stravitz et al (11) studied a group of 72 adult patients with ID-FHF and proposed a histological classification that pointed at centrilobular disease (MHN4 and MHN5) as a feature of AI-FHF. In our study we could not demonstrate a greater prevalence of MHN4–5 in patients with AI-FHF compared with those with ID-FHF. Histological evaluation was more helpful when scored according to SDC criteria, although we were unable to show a clearcut difference between G1 and G2 patients, both for the SDC and for the MHN histology classifications. Histology can differentiate well AI-FHF when the features are typical or absent according to the SDC criteria, whereas it has low diagnostic power if the pattern is less striking, as occurred in many of our patients, questioning the utility of performing a dangerous liver biopsy in the presence of severe coagulopathy and encephalopathy. AIH tends to develop and present later in childhood than other paediatric liver disorders, which often have a genetic cause (35,36). Several authors have regarded AIH as a rare cause of FHF in children
