INTRODUCTION
Autoimmune liver disease (AILD) is a broad term that includes multiple closely related etiologies such as autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), overlap syndrome (AIH-PBC, AIH-primary sclerosing cholangitis (PSC)), IgG4 cholangitis, etc., AIH is diagnosed by a combination of histological, serological, and biochemical parameters as per simplified criteria.[1,2] The various serological markers include the presence of autoimmune markers such as ANA, anti-smooth muscle antibody (ASMA), LKM1, and LC1 along with elevated serum immunoglobulin G (IgG) levels. While serum IgG level is a diagnostic marker of AIH, elevated levels can also be seen in advanced stages of chronic liver diseases irrespective of the etiology.[3]
AIH is traditionally associated with raised IgG levels; however, the cut-off value that helps in differentiating it from other autoimmune diseases such as PBC, or from a non-immune mediated etiology such as non-alcoholic steatohepatitis (NASH), is highly debated. The original International Autoimmune Hepatitis Group (IAIHG) criteria for diagnosing AIH gave a scoring system that would provide a score of 1 for elevation of IgG levels by 1 to 1.5 times, a score of 2 for elevation by 1.5 to 2 times, and a score of 3 for elevation by more than 2 times.[4,5] However, this was replaced by the simplified criteria which recommend a score of 1 for elevation by 1 to 1.1 times the upper limit of normal (ULN) and a score of 2 for more than 1.1 times the upper limit.[1,6] On the other hand, the Paris criteria for diagnosing overlap AIH-PBC, recommends a cut-off IgG value of >2 times ULN to diagnose the AIH component of overlap.[7] This brings to us two important questions:
“What is the ideal cut-off value of IgG to differentiate AIH from other pathologies?”
“Is there any significance of these different cut-off values mentioned in literature?”
To the best of our knowledge, there are no studies in the literature that assess the significance of serum IgG levels and the need for an ideal cut-off for the exact diagnosis of AIH which is crucial for patient management. Hence, we aimed to study the association of elevated IgG levels with the severity of AIH and to find out the ideal cut-off of IgG in differentiating AIH from other autoimmune and non-autoimmune pathologies.
MATERIALS AND METHODS
Study design
A cross-sectional study was performed among patients of AILDs who presented to our tertiary referral center between March 2010 and March 2020. The study was approved by the Institute’s ethics committee (IEC 2021/83/MA22).
Study population
Data of all histopathologically confirmed AILD cases were collected which includes demographic parameters, liver function tests, autoimmune markers, and serum IgG levels.
All cases with histopathological diagnosis of AILDs such as AIH, PBC, and overlap (AIH-PBC) fulfilling standard diagnostic criteria with an adequate biopsy length of >1 cm or >10 portal tracts were all included. Non-cirrhotic NASH cases were also included as controls in which serum IgG levels were performed for clinical suspicion of AIH. Cases with inadequate biopsy length (<1 cm), inadequate laboratory details, and other alternative diagnoses were all excluded. Also, only pre-treatment biopsies were included.
AIH was diagnosed based on the latest (2008) simplified criteria which was based upon serum IgG levels, ANA, SMA, LKM, SLA, presence of viral hepatitis, and liver histology. The histological features of AIH described are interface hepatitis, lymphocytic/lymphoplasmacytic infiltrates in portal tracts and extending into the lobule, emperipolesis (active penetration by one cell into and through a larger cell), and hepatic rosette formation. All these features must be present to make a diagnosis of “Typical AIH” while the absence of any of these features but a picture of chronic hepatitis and lymphocyte infiltration is considered as “Compatible with AIH.” Histology was considered “Atypical” when signs of another diagnosis such as steatohepatitis were noted.[1,2]
On the other hand, the criteria for diagnosis of PBC includes a cholestatic serum enzyme, elevated serum IgM, presence of AMA (>1:40 titer)/AMA-M2 with histological features of PBC (intrahepatic small bile duct injury-florid bile duct lesion, granulomatous cholangitis with ductopenia, and cholestasis).[8,9]
AIH-PBC overlap syndrome is diagnosed by Paris criteria: two or three criteria of PBC 1) Alkaline phosphatase (ALP) >2 times ULN or gamma-glutamyl transferase (GGT) >5 times ULN, 2) AMA Positive, and 3) florid bile duct lesions as well as AIH; 1) alanine transaminase (ALT) >5 times ULN, 2) IgG >2 times ULN or ASMA positivity, and 3) portal and periportal lymphocytic inflammation with moderate or severe periportal lymphocytic interface hepatitis.[7,10]
A total of 17664 liver biopsies were received in the Department of Pathology between March 2010 and March 2020. After excluding the follow-up biopsies and cases with inadequate details, we had 630 biopsies with the same number of patients with histopathological diagnoses of AILD such as AIH, PBC, and overlap (AIH-PBC).
Histology
The study material comprised serial step sections of liver biopsy and explants stained with hematoxylin and eosin, orcein, and Masson’s trichrome stain. Cytokeratin (CK7) immunohistochemistry was done in cases whenever needed to confirm the absence of ductal profiles. Two pathologists independently reviewed the liver biopsies and reassessed 18 histopathological parameters in all the cases and graded them as in the following table. Any discrepancy in interpretation was resolved by simultaneous viewing on a multiheaded microscope in the presence of a third pathologist [Table 1].
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Table 1: Various histological parameters and their grading
In cases of AIH, modified Ishak’s Histological Activity Index (HAI) and Ishak’s staging were also recorded.[11,12]
Non-cirrhotic NASH cases, in which IgG levels were done as a part of a work-up to exclude AIH were included as controls and this group comprised 209 cases.
Statistical analysis
The quantitative data were expressed as mean or median values and the qualitative data were enumerated as frequencies and percentages. Comparison between the groups was carried out by Fisher’s exact test or Pearson’s Chi-square test for categorical variables. When analyzing continuous data between more than two groups, analysis of variance (ANOVA) was employed for parametric data while the Kruskal–Wallis test was employed for non-parametric data. A P value of <0.05 was considered to indicate a significant difference. All data were recorded and analyzed using the SPSS v20.0 software package (SPSS Inc., Chicago, Illinois, USA).
RESULTS
After exclusion, a total of 630 patients of AILD were included in the present study. Out of these, 455 patients formed the AIH group, 97 formed the PBC group, and 78 patients formed the overlap (AIH-PBC) group. The median age of patients in the AIH group was 45 years as against 48 years and 48.5 years in the PBC group and overlap (AIH-PBC) group, respectively. Females constituted 84% of the total in the PBC group, while the AIH group had 60.5% and the overlap group had 76% females.
The median IgG level was 22.4, 17.9, and 20 in the AIH, PBC, and overlap (AIH-PBC) groups, respectively. The IgG level was further subdivided into four different classes as depicted in Table 2.
Table 2: Different classes of IgG
As it is evident from Table 2, most patients with IgG elevation of >2 times ULN belonged to the AIH group. On the other hand, 79.5% of patients in the overlap group had IgG levels below two times ULN but still they qualified for overlap syndrome based on other parameters. Hence, the criterion of serum IgG levels >2 times the upper limit for diagnosing AIH component of overlap in Paris Criteria has too many false negatives and mandates revision.
We tried to deduce the significance of this cut-off value of >2 times ULN in AIH patients by studying the differences in their laboratory, serological, and histological parameters. Of the 455 patients in the AIH group, two subgroups were identified—155 patients belonged to the >2 times ULN subgroup (Subgroup A) while the rest formed the <2 times ULN subgroup (Subgroup B). Patients in subgroup A had severe hepatitis as demonstrated by significantly higher aspartate transaminase (AST), alanine transaminase (ALT), ALP, and bilirubin levels as compared to subgroup B. This is also correlated with the findings in the histological picture. Subgroup A had significantly severe portal inflammation, interface activity, lobular inflammation, ductular proliferation, necrosis, Ishak’s HAI index, and more portal and lobular plasma cells. However, the difference in Ishak’s stage between the two subgroups is not statistically significant [Table 3, Figure 1].
Table 3: Comparing biochemical and histopathological parameters among AIH patients
Figure 1: Higher necro inflammatory activity in patients with serum IgG levels >2 ULN. (a) Moderate degree of portal inflammation with prominent plasma cells (H and E stain-10×). (b) Moderate interface activity (H and E stain 10×) (c) Prominent lobular plasma cell aggregates (H and E stain 20×). (d) Bridging necrosis (Hematoxylin and eosin stain 4×) (e and f) Bridging necrosis (Masson trichrome 4×-pale blue areas of necrosis and Orcein stain 4×-unstained areas of necrosis)
Another inference from Table 2 is that 42% (87 patients) of NASH patients had IgG levels more than 1.1 times ULN while 69% (43 patients) of patients in the PBC group had such levels. Thus, it is evident that this cut-off value of IgG will have too many false positives, i.e., it lacks specificity in differentiating AIH from either NASH or PBC. An ideal cut-off value should have acceptable sensitivity and specificity and should be uniformly applicable to diagnose both isolated AIH as well as AIH components of overlap syndrome.
To find out the ideal serum IgG cut-off value, patients with advanced stages of liver diseases and pediatric AIH were excluded. Since, elevated IgG levels are associated with advanced stages of liver diseases, cases with cirrhosis have been excluded to look for the exact IgG cut-off levels. The percentage of patients with cirrhosis was 27.4% (125 cases), 36% (35 cases) in the PBC group, and 28% (22 cases) in the overlap (AIH-PBC) group. Even pediatric AILD cases (70 cases) were excluded. Hence, after excluding cirrhotic and pediatric patients, we had 378 cases of AILDs for further analysis [Figure 1].
To identify this cut-off, a receiver operating characteristic (ROC) curve was plotted for different IgG cut-off values to differentiate AIH vs NASH [Figure 2]. It was found that in our study population, an IgG cut-off value of >1.3 times ULN has the maximum area under the curve (AUC). This cut-off value has a sensitivity of 78.9% and a specificity of 85.7%. The same cut-off was applied to differentiate overlap vs NASH. The specificity was 85.7% while sensitivity was 66%.
Figure 2: ROC curve of IgG cut-off values comparing AIH group vs NASH group
DISCUSSION AND CONCLUSION
The distribution of AIH, PBC, and Overlap AIH-PBC syndrome in our study cohort is 72%, 16%, and 12%, respectively. Poupon et al.[13] had reported a prevalence of 14% overlap syndrome in a study of 282 patients while Chazouillères et al.[14] had reported an incidence of 9%. Among the 17664 liver biopsy samples screened in our study, 1060 samples (6%) belonged to patients with AILD. Thus, it is evident that AILD is a relatively rare disorder among patients undergoing liver biopsies in our study cohort. Tanaka et al.[15] had reported that AILD is rarer in the Asia-Pacific region compared to the Western population. To date, this is the largest cohort of AILD patients reported from a single center in India. Previously, Amarapurkar et al.[16] had reported a cohort of 181 patients from India, with AIH prevalence of 1.3% and 8.4% among all liver disease patients and CLD patients, respectively. In the absence of population-based incidence data, our study findings are closely reflective of the true incidence among the Indian population.
While the incidence of true overlap syndrome reported varies greatly in literature from 2% to 19%, this difference is not entirely due to true variation in different study populations. Some of the differences in incidence may be attributed to the variable diagnostic criteria employed by different authors. In 2011, IAIHG gave a position statement where it mentioned that “standardized definitions of ‘overlap syndromes’ are lacking” and they cautioned against the use of IAIHG criteria for diagnosing “Overlap syndromes.”[17] Liu et al.[18] had compared the utility of three different criteria in the same PBC cohort and found that the incidence of overlap syndrome varied greatly from 3% to 20% depending on the criteria employed. Hence, a uniform, acceptable diagnostic criterion for diagnosis is the need of the hour to understand the true incidence of overlap syndrome.
One major difference between IAIHG criteria, simplified criteria, and Paris criteria[7] is the different serum IgG cut-off values proposed for diagnosing AIH. Serum IgG level is characteristically elevated in patients with AIH and it is an important marker for the diagnosis as well. Our study shows that patients with AIH had the highest mean levels of IgG compared to PBC and NASH cases. A novel finding in our study is that elevation of serum IgG beyond two times ULN in AIH patients at presentation is associated with severe hepatitis as reflected in both biochemical as well as histopathological parameters. Whether this elevation in serum IgG has a causal role or is a consequence of severe hepatitis needs further evaluation. In patients with chronic hepatitis C, Maruyama et al.[19] had reported that elevated serum IgG level had a strong correlation with disease severity as assessed by HAI score, grading score, and staging score as well as treatment outcome. Li et al. studied that lower levels of serum IgG were associated with lesser fibrosis and predicted biochemical and histological remission in patients with AIH.[20] To the best of our knowledge, ours is the first study to report an association of higher levels of serum IgG with disease severity in AIH patients. Prospective studies are warranted to confirm this association and to assess the utility of this cut-off value in predicting treatment response.
While the serum IgG cut-off value for diagnosis of AIH component in Paris criteria lacks sensitivity, the one proposed in simplified criteria lacks specificity. Zhang et al.[21] had reported a sensitivity of 59% for Paris criteria in diagnosing overlap syndrome. This led them to develop their own criteria using four groups of parameters—biochemical, immunological, histologic, and others. They had defined a score of ≥21 points as “Definite OS” while <19 is “Rejected.” Scores 19 and 20 are “Probable OS.” However, such exhaustive scoring systems are unlikely to find universal applicability. Our study had employed an ROC curve to find the ideal cut-off value against a control group of non-cirrhotic NASH patients. For this analysis, we excluded cirrhotic AILD patients to avoid confounding effects. We found that the maximum AUC was at 1.3 times ULN and we propose this as a new universal IgG cut-off value for diagnosing AIH.
Our study has certain limitations. By virtue of its retrospective design, inadequate data resulted in the exclusion of some eligible study subjects. Moreover, some AILD patients (especially those with PBC) who presented to our institute may not have been subjected to liver biopsy. Nevertheless, our study results can judiciously answer our research questions despite these shortcomings.
To conclude, serum IgG level is a useful marker for diagnosis and severity of AILD. The cut-off values proposed in IAIHG, simplified and Paris criteria need to be revised. Our study findings suggest that IgG levels >2 times ULN in AIH patients are associated with severe hepatitis as per serological parameters. This finding is novel as this correlation has never been reported in the literature. Moreover, the severity of the disease process is also reflected in the histological picture. Our study results further demonstrate that the IgG cut-off value of 1.3 times ULN has the maximum sensitivity and specificity for diagnosing isolated AIH as well as AIH component of overlap AIH-PBC.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgements
Prof. (Dr) SK Sarin, Senior Professor (Dept. of Hepatology) and Director, Institute of Liver and Biliary Sciences, Delhi, for constant guidance and support.
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