If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Corresponding author: Head of the Department of Medicine and the Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, Full Member of the European Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Mezourlo area, Larissa 41110, Greece
Department of Medicine and Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, General University Hospital of Larissa, Larissa, GreeceEuropean Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Larissa, Greece
Department of Medicine and Research Laboratory of Internal Medicine, National Expertise Center of Greece in Autoimmune Liver Diseases, General University Hospital of Larissa, Larissa, GreeceEuropean Reference Network on Hepatological Diseases (ERN RARE-LIVER), General University Hospital of Larissa, Larissa, Greece
There is not a single specific laboratory marker to diagnose or exclude AIH.
•
Therefore, diagnosis of AIH is in most cases challenging for physicians.
•
Antibodies detection is mandatory even though not pathognomonic for AIH diagnosis.
•
Assessment of autoimmune serology in strict adherence to guidelines is essential.
•
IgG increase and autoimmunity background support an in-depth serological assessment.
Abstract
Diagnosis of autoimmune hepatitis (AIH) is in most cases challenging for clinicians as there is not a single specific laboratory or histological marker to diagnose or exclude the presence of the disease. The clinical spectrum of AIH varies from completely asymptomatic to acute-severe or even rarely fulminant hepatic failure, while everybody can be affected irrespective of age, gender, and ethnicity. The old revised and the newer simplified diagnostic scores have been established by the International Autoimmune Hepatitis Group (IAIHG) in 1999 and 2008, respectively, which are based on several clinical, laboratory and histological parameters. Additionally, a thorough differential diagnosis from other diseases mimicking AIH is absolutely indicated. In this context, autoantibodies detection in patients with suspected AIH is mandatory -even though not pathognomonic- not only for AIH diagnosis but furthermore, for AIH classification (AIH-type 1 and AIH-type 2). Although autoimmune serology can be supportive of AIH diagnosis in ≥95% of cases if testing has been performed according to the IAIHG guidelines, this is not the case under real-life circumstances in routine clinical laboratories. Clinicians should be careful both for the importance of the required testing and how to interpret the results and therefore, they should communicate and discuss with the laboratory personnel to achieve the maximum benefit for the patient. Herein, a detailed and updated review of the diagnostic work-up for AIH diagnosis under real-life conditions is given to minimize the underestimation and misdiagnosis of AIH which can result in progression of the disease and unfavourable outcomes.
The diagnosis of autoimmune hepatitis (AIH) is based on a combination of clinical, laboratory and histological findings in patients with unexplained acute or chronic hepatitis [
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
]. As in most autoimmune diseases, AIH is characterized by female predominance while every subject can be affected irrespective of age, gender, and ethnicity [
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
The international autoimmune hepatitis group (IAIHG) has established diagnostic scores to help for a timely and prompt diagnosis of the disease namely, the revised and the simplified score in 1999 and 2008, respectively [
]. The latter seems very easy and friendly for use in everyday clinical practice as only four parameters have been included namely, autoantibodies detection, gamma-globulins or immunoglobulin G (IgG) serum levels, histopathology of the liver, and seronegativity for viral hepatitis markers (Supplementary Table 1) [
]. However, even though the abovementioned simplified criteria represent a good tool for daily clinical practice still, there is not a diagnostic ''gold standard'' and therefore, the clinicians must regard any diagnostic score only as an aid to AIH diagnosis and the criteria should be used alongside clinical judgement [
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
It should be noted also, that recently many doubts have been raised concerning the significance of emperipolesis and hepatocyte rosettes as typical characteristics of AIH at the histological level [
]. In this regard, the International AIH Pathology Group very recently published a consensus report which proposed a homogenous approach for AIH diagnosis based on liver histology [
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
]. However, up to the present, this consensus lacks external validation and therefore, cannot be used safely in everyday clinical practice by replacing that of the simplified criteria. Besides, the consensus opinion is that even though hepatocyte rosettes and emperipolesis are not specific histological characteristics of AIH, they can be reported as surrogate markers of AIH severity [
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
]. Detailed description of the new proposed recommendations for the histological criteria of AIH is beyond the scope of this review (for further reading see ref. [
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
According to the autoantibodies detected, AIH is classified as AIH-type 1 (AIH-1) or AIH-type 2 (AIH-2). Patients with AIH-1 have detectable anti–nuclear autoantibodies (ANA) and/or smooth muscle autoantibodies (SMA). Patients with AIH-2 have detectable anti–liver kidney microsomal type-1 (anti-LKM1) or rarely anti–liver kidney microsomal type-3 (anti-LKM3), and/or anti–liver cytosol type-1 (anti-LC1) antibodies [
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
]. This review aims to provide an updated roadmap for the performance of autoimmune serology to facilitate physicians both to be aware of the importance of this testing but also to be efficient to interpretate the results.
2. Autoantibodies detection
2.1 Anti-nuclear (ANA) and smooth muscle antibodies (SMA)
According to the current guidelines, ANA should be detected in first-line screening by indirect immunofluorescence (IIF) on fresh-frozen rodent stomach, liver, and kidney tissue substrates (Fig. 1, Fig. 2) [
]. Up to the present, several target-autoantigens of ANA have been identified in AIH patients, including single- and double-stranded DNA (dsDNA), chromatin, histones, centromere, ribonucleoproteins, and cyclin A (Table 2). However, neither the staining pattern by using the human larynx epithelioma cancer (HEp-2) cell lines as substrate, nor the identification of the target-autoantigens have any known specific clinical implication in patients with AIH.
Fig. 1Antinuclear antibodies by indirect immunofluorescence on (A) HEp2 cells showing a homogeneous nuclear staining and (B) fresh rodent liver section with staining of the nuclei of hepatocytes. Original magnification × 40.
Fig. 2Abbreviations are same as in the text. Diagnostic algorithm of investigation in patients with acute or chronic hepatitis of unknown cause. Autoantibodies can be detected in more than 95% of patients if testing performed according to the guidelines. Under real life conditions, in cases with acute severe AIH (jaundice, international normalized ratio≥1.5, no hepatic encephalopathy, no previously recognized liver disease) a diagnostic trial with oral or intravenous corticosteroids is rather justified before obtaining the results of autoimmune serology and liver biopsy, as the abovementioned diagnostic algorithm is somehow time consuming.
*IgG levels can also be within normal limits in about 39% of acute severe cases. **ANA and SMA can also be evaluated by IIF on HEp2 cells or by ELISAs (for details and rules see text and Table 3) [
Table 2Significance of antibodies in autoimmune hepatitis (AIH).
Antibody
Target-autoantigens/Detection Methods
Clinical significance
ANA
Histones, chromatin, ribonucleoproteins; single- and double-stranded DNA, cyclin A, centromere; undefined antigens 20–30%; IIF on multi-organ rodent tissue substrates*
AIH-1 but not specific; Rare in AIH-2
SMA
Filamentous actin, desmin, vimentin; Undefined antigens in 20%; IIF on multi-organ rodent tissue substrates*
AIH-1 particularly in combi-nation with ANA; VG/VGT patterns highly specific; Rare in AIH-2
Anti-LKM1
Cytochrome P450 2D6 (molecular weight: 50 kDa); IIF on multi-organ rodent tissue substrates; also, by ELISA or western blot
Highly specific for AIH-2 (absent in AIH-1); detectable in HCV infection (10%)
Anti-LKM3
UGT1 (molecular weight: 55 kDa); IIF on multi-organ rodent tissue substrates or by western blot
Specific antibody for AIH-2 but rare; present in up to 13% of HDV infection
Anti-LC1
FTCD (molecular weight: 58–62 kDa); IIF on multi-organ rodent tissue substrates; also, by immunodiffusion, ELISA, or western blot (very important in patients with coexistence of anti-LKM1 by IIF)
Liver specific antibody for AIH-2 (absent in AIH-1 and rare in HCV); usually coexists with anti-LKM1; can be the only marker (10% of AIH-2 cases)
Anti-SLA/LP
Synthase (S) converting O-phosphoseryl-tRNA (Sep) to selenocysteinyl-tRNA (Sec) (molecular weight: 50 kDa); ELISA or western blot
Highly specific for AIH-1 (15–30% of patients; specifi-city: 99%); rare in AIH-2; coexists with anti-Ro52 anti-bodies (77–98% of cases); need for permanent treatment
pANCA/ pANNA
Unknown target-autoantigen; IIF on fixed granulocytes
Exclusively in AIH-1 (60–96%); very few cases with isolated detection; also in PSC, IBD and ASC patients
PBC-specific ANA
Nuclear body speckled 100 kDa (sp100) and 210 kDa glycoprotein (gp210) as main target autoantigens of MND and RLM patterns by IIF on HEp2 cells; also, by ELISAs or western blot
Very specific for PBC (up to 65% of PBC patients by using specific IgG subclasses as secondary antibody on HEp2 vs. 15–20% by the common anti-total IgG use); associate with PBC severity
Anti-α-actinin
α-actinin (F-actin cross-linking protein); investigational serologic marker; ELISA or western blot
Common in SLE and AIH-1; 42% of AIH-1 and in 2/3 of anti-F-actin positive (double reactivity is associated with severe AIH, insufficient res-ponse, and relapse episodes)
Anti-dsDNA
dsDNA usually by ELISAs or IIF on Crithidia luciliae (higher specificity than molecular-based assays)
Highly specific for SLE and AIH (30% of AIH and up to 60% of AIH/PBC variant); SLE misdiagnosis instead of concurrent AIH or AIH alone is not uncommon with po-tential catastrophic consequ-ences for the patients
AMA
E2 subunits of the 2-OADC; IIF on multi-organ rodent tissue substrates; also, by specific ELISAs or western blot
Laboratory hallmark of PBC but also in 5–10% of typical AIH cases (significance unknown); similar manage-ment as in AMA-negative AIH patients
Abbreviations are same as in the text. *For updates regarding their detection by IIF on HEp2-cells or ELISAs see Table 3 and text; CYP2D6, cytochrome P450 2D6; HCV, hepatitis C virus; UGT1, family 1 of uridine diphosphate glucuronosyl-transferases; HDV, hepatitis D virus; FTCD, formiminotransferase cyclodeaminase; IBD, inflammatory bowel disease; ASC, autoimmune sclerosing cholangitis; PBC, primary biliary cholangitis; MND, multiple nuclear dots; RLM, rim like membranous; 2-OADC, 2-oxo-acid dehydrogenase complexes
Even though ANA are currently the most sensitive marker of AIH, they lack specificity as they are present in a variety of liver diseases, including fatty liver disease, drug-induced liver injury (DILI), viral hepatitis, Wilson's disease, and alcoholic liver disease [
As in ANA, the detection of SMA is recommended by IIF on fresh-frozen triple rodent substrates. Τhey stain the lamina propria and muscularis mucosae of the stomach (Fig. 3A) and arterial walls of the liver, while three different IIF patterns characterize SMA on rodent kidney substrate: (1) V-pattern (arterial vessels), (2) VG-pattern (arterial vessels and mesangium of renal glomeruli), and (3) VGT-pattern (arterial vessels, glomeruli and intracellular fibrils in renal tubule) (Fig. 3B) [
]. SMA target-autoantigens include structures of the cytoskeleton, mainly the filamentous actin (anti-F-actin which is present in 80% of AIH patients with SMA of VGT-pattern), but also vimentin, tubulin desmin, and troponin (Table 2) [
Fig. 3Smooth muscle antibodies on fresh rodent tissue sections by indirect immunofluorescence. (A) Staining of the smooth muscle fibers of lamina propria and muscularis mucosae of the stomach and (B) Staining of the smooth muscle fibers within the arterial vessels and glomeruli of the kidney. Original magnification × 40.
According to the current criteria for AIH, IIF titers ≥1:40 in adults and ≥1:20 in children are considered positive for ANA and SMA during the investigation of liver diseases [
]. Of interest, SMA and ANA are only useful for diagnosing AIH, whereas they are not associated with prognosis. SMA are typically found in conjunction with ANA in about 50% of patients, whereas isolated SMA are detected in approximately 35% and ANA alone in 15% of cases [
2.2 Antibodies against soluble liver antigens/liver pancreas (anti-SLA/LP)
Anti‐SLA/LP are detected by enzyme-linked immunosorbent assay (ELISA), immunoblot or radioligand assays but not with conventional IIF in 15%‐30% of AIH patients and according to the guidelines should be investigated during the first-line screening (Fig. 2) [
]. The target-autoantigen of anti-SLA/LP has been identified as a synthase (S) converting O-phosphoseryl-tRNA (Sep) to selenocysteinyl-tRNA (Sec) (Table 2) [
Meta-analysis: diagnostic accuracy of antinuclear antibodies, smooth muscle antibodies and antibodies to a soluble liver antigen/liver pancreas in autoimmune hepatitis.
]. Recent studies in large cohorts of patients have shown that seropositivity for anti-SLA/LP was not associated with the clinical, serological, biochemical, or histological features of AIH patients [
]. However, anti-SLA/LP-positive patients have lower sustained rates of biochemical response after complete cessation of treatment suggesting that rather permanent immunosuppression is needed in this subgroup of AIH patients [
Another important point is that anti-SLA/LP is associated with the concurrent detection of autoantibodies against to the ribonucleoprotein/Sjogren's syndrome A antigen (anti-Ro/SSA) [
]. Indeed 77–98% of anti-SLA/LP-positive European and North American AIH patients have also anti-Ro52 antibodies which is not a result of cross-reactivity [
]. From the clinical and diagnostic points of view, the abovementioned findings seem very important as the clinician could suspect the presence of underlying AIH in a patient with unexplained hepatitis and anti-Ro52 antibodies by searching for the potential concurrent presence of anti-SLA/LP in case this testing has not been done (Table 2).
2.3 Anti-neutrophil cytoplasmic antibodies (ANCA)
These autoantibodies have been originally described by IIF on fixed granulocytes in patients with granulomatosis with polyangiitis (typically associated with diffuse cytoplasmic granular fluorescence; cANCA; major target-autoantigen: proteinase 3; Fig. 4A) or microscopic polyangiitis and eosinophilic granulomatosis (typically associated with perinuclear staining pattern; pANCA; major target-autoantigen: myeloperoxidase) [
Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK).
]. The latter staining pattern is an artifact because of the ethanol fixation of granulocytes, which results in the migration of positive cytoplasmic proteins to the negative nuclear cell membrane. For these reasons, both formalin and ethanol fixed granulocytes should be used in ANCA investigation. If the pattern of staining is not affected by ethanol fixation, the result should be given as “atypical” pANCA (Fig. 4B), or with the more appropriate term “perinuclear antineutrophil nuclear antibody” (pANNA; positive cut-off titre 1:20; Fig. 4C; Table 3).
Fig. 4Anti-neutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence on ethanol fixed human granulocytes: (A) Granular cytoplasmic staining (cANCA); (B) “Atypical” perinuclear staining (pANCA); (C) Anti-neutrophil nuclear antibody staining (ANNA). Original magnification x 40.
Table 3Update of the simplified diagnostic criteria of the International AIH Group [adapted from 60].
Feature
Cut-off
Points1
ANA or SMA/anti-F-actin
Positive2
1
ANA or SMA/anti-F-actin
Strongly positive3
or anti-LKM
≥1:40
2
or anti-SLA/LP
Positive
IgG
>Upper limit of normal
1
>1.1x upper limit of normal
2
Liver histology (with evidence of hepatitis)
Compatible with AIH
1
Typical AIH
2
Absence of viral hepatitis
Yes
2
≥6: probable AIH
≥7: definite AIH
Abbreviations are same as in the text. 1Addition of points achieved (maximum 2 points for autoantibodies). 2Indirect immunofluorescence: ≥1:40 when assessed on tissue sections; ≥1:80 or 1:160 for ANA when assessed on HEp-2 cells, depending on local standards. ELISA with locally established cut-offs. 3Indirect immunofluorescence: ≥1:80 when assessed on tissue sections; ≥1:160 or 1:320 for ANA when assessed on HEp-2 cells. ELISA with cut-offs established locally; Important note: If ELISA-based autoantibody assessment is negative despite clinical suspicion of autoimmune hepatitis, indirect immunofluorescence should be performed in addition.
Searching for specific autoantibodies against proteinase 3 and myeloperoxidase by molecular-based assays has not any sense in the investigation of an index patient with suspected AIH and detectable pANCA/ANNA. Neither the determination of antigenic specificities of pANCA/ANNA seems to be important as the clinical relevance of such investigation in AIH patients is obscure [
Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK).
]. However, very few AIH-1 patients have isolated pANCA/ANNA and therefore, this autoantibody should be tested only in patients who are negative for ANA, SMA, and anti-SLA/LP. pANCA/ANNA has also been reported frequently in other immune-mediated diseases such as, primary sclerosing cholangitis (PSC), inflammatory bowel disease and autoimmune sclerosing cholangitis a specific AIH/PSC variant in children (Table 2) [
]. Anti-LKM1 and less commonly anti-LKM3 define AIH-2, whereas anti-LKM2 antibodies are associated with DILI due to tienilic acid and never with AIH (Table 2) [
]. On fresh rodent tissue sections, anti-LKM1 shows by IIF a restricted staining of the third portion of the proximal renal tubules (Fig. 5A). Besides, anti-LKM1 stain the cytoplasm of the complete liver lobule (Fig. 5B). Anti-LKM1 are easily distinguished from anti-mitochondrial antibodies (AMA) as the latter antibodies stain both the proximal and distal renal tubules. In any case, if there are problems regarding the discrimination between AMA and anti-LKM by IIF, these should be resolved by using complementary molecular-based assays.
Fig. 5Liver kidney microsomal antibodies type 1 (anti-LKM1) and liver cytosol type 1 antibodies (anti-LC1) on fresh rodent tissue sections by indirect immunofluorescence. Anti-LKM1 shows (A) a restricted staining of the P3 segment of the proximal tubules sparing the distal tubules and (B) a diffuse staining of the cytoplasm of hepatocytes in the entire liver lobule. (C) Anti-LC1 exhibits a pattern of cytoplasmic staining of the periportal hepatocytes while sparing the area around the central veins. Original magnification × 40.
The main target-autoantigen of anti-LKM1 is the cytochrome P450 2D6, which allowed the development of specific molecular-based assays for anti-LKM1 detection, such as ELISA and immunoblot. Interestingly, about 10% of patients with chronic hepatitis C but not with hepatitis B, have detectable anti-LKM1 [
]. In about 10% of AIH-2 patients, anti-LC1 autoantibodies are detected solely, so it is mandatory to be included at first-line screening in the diagnostic algorithm of suspected cases (Fig. 2) [
]. Anti-LC1 exhibits by IIF a cytoplasmic staining pattern of the periportal hepatocytes on fresh rodent tissue sections, sparing typically the area around the central veins (Fig. 5C) [
]. As a result, in cases of anti-LC1 coexistence with anti-LKM1 antibodies, IIF fails to detect them because anti-LKM1 stains the cytoplasm of hepatocytes in the entire liver lobule (Fig. 5B). Therefore, additional methods like immunodiffusion, ELISA or immunoblot are required [
Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques.
]. The formiminotransferase cyclodeaminase, a liver enzyme involved in folate metabolism, has already been documented as the molecular target of anti-LC1 [
3. Diagnostic application in patients with deranged liver enzymes of unknown aetiology
The investigation of autoantibodies remains the hallmark for AIH diagnosis, even though they are not pathognomonic and cannot support a definite diagnosis on their own. A step-by-step serological investigation which should be applied in patients with deranged liver enzymes of unexplained cause is shown in Fig. 2. It should be noted however, that in cases with acute severe form of the disease (jaundice, international normalized ratio≥1.5, no hepatic encephalopathy, no previously recognized liver disease) a diagnostic trial with oral or intravenous corticosteroids is rather justified before obtaining the results of autoimmune serology and liver biopsy [
Prompt initiation of high-dose i.v. corticosteroids seem to prevent progression to liver failure in patients with original acute severe autoimmune hepatitis.
]. Furthermore, it should be clear that in cases of suspected AIH with acute liver failure (which includes by definition the presence of hepatic encephalopathy), the patients should be immediately listed for liver transplantation even though an autoimmune serology testing could be in process [
ANA, SMA, anti-LKM-1 and anti-LC1 antibodies must be investigated first by IIF on fresh-frozen substrates from combined rodent liver, stomach, and kidney sections ideally before treatment initiation as immunosuppression may affect the results. In parallel, investigation for anti-SLA/LP antibodies by molecular-based assays (ELISAs and/or western blot) should be done (Fig. 2). Reactivity for one or more of these autoantibodies, in conjunction with the appropriate clinical background, suggests proceeding to liver biopsy, as AIH is highly likely [
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
]. Absence of reactivity besides clinical suspicion, raises the justification for additional specific searching and repeated investigation for ANA, SMA, anti-LKM-1, anti-LC1 and anti-SLA/LP and non-standard autoantibodies in a reference laboratory (Fig. 2). Insistence for second testing is further supported if there are concurrent extra-hepatic autoimmune diseases in the patient or her/his first-degree relatives [
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
]. Absence of reactivity after the second investigation means that AIH is very unlikely and other diagnosis should be considered or seronegative AIH is present. It is worthy to state here, that the above thorough serological investigation could be engaged even for patients with other liver disorders if there is distinct IgG elevation and an autoimmune extra-hepatic background because coexistence of AIH with another liver condition cannot be excluded [
The routine clinical laboratories should adhere to the guidelines both regarding the assays they use as well as the cut-off they consider for positivity and this information should be provided clearly to the clinicians to assist them achieving the appropriate interpretation of the results [
]. In this context, it is pivotal the laboratory to report all renal reactivity patterns of SMA, as SMA-VG/VGT patterns have the highest specificity for AIH diagnosis.
3.1 Common difficult clinical questions
My lab does not perform IIF testing on triple rodent substrates but relies on IIF on HEp-2 cells and/or ELISAs for ANA and SMA detection. How shall I manage this difficulty?
It should be emphasized that the original simplified score (Supplementary Table 1) does not account for ANA or SMA detection by IIF on the HEp-2 cell lines or by using ELISAs even though, these assays are indeed the preferable ways of screening in many routine laboratories because they are much easier and more friendly in everyday use. However, it is not clear how the results by using HEp2 cells and ELISAs may be converted into the simplified diagnostic score [
]. This happens because most of these assays have been developed mainly for the investigation of patients with autoimmune rheumatic diseases and therefore, they utilize molecularly defined antigens only as substrate and not whole nuclear extracts which is very important for the investigation of suspected cases of AIH [
Moreover, the use of F-actin ELISA for SMA detection can lose about 20% of cases, as SMA do not target F-actin exclusively, and therefore, anti-F-actin molecular assays should only be used in conjunction with IIF [
]. To sum up, clinicians should be very careful and aware of these arbitrary assumptions of the laboratories as they may have dramatic consequences for the patients with suspected AIH.
Of note, the IAIHG recently aimed to address these issues by comparing the performance of IIF on HEp-2 cells and tissue sections for the detection of ANA and SMA in AIH patients and controls from three European centres [
]. It was concluded that, if ANA titres on HEp-2 cells adjust to higher thresholds (1/160 for positivity and 1/320 for strong positivity; Table 3), then this substrate could be used as a reliable alternative assay for ANA detection [
Regarding the various ELISA kits, it was shown that they can also be used as alternative assays for ANA and SMA but for ANA the use of total HEp-2 nuclear extracts should be applied in order to account for unrecognised nuclear autoantigens in addition to molecularly defined antigens, whereas for both ANA and SMA ELISAs there is a need for locally established cut-offs in each laboratory as the cut-offs of these commercial ELISAs have not been validated globally [
]. The proposed updated simplified score which, however, needs validation is shown in Table 3.
A special mention on screening by HEp-2 cells is needed for two specific ANA patterns namely, the multiple-nuclear dot and rim-like membranous patterns which are commonly seen in PBC patients (Fig. 6; Table 2) [
European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
Autoantibody status and histological variables influence biochemical response to treatment and long-term outcomes in Japanese patients with primary biliary cirrhosis.
]. The presence of PBC-specific ANA in AIH patients in conjunction with a cholestatic biochemical profile may suggest the diagnosis of AIH/PBC variant and therefore, liver biopsy should be carefully evaluated in these cases [
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
Fig. 6Antinuclear antibodies specific for primary biliary cholangitis. Detection by indirect immunofluorescence on HEp-2 cells. (A) Multiple-nuclear dot staining pattern of 3–20 dots of variable size in the nucleus; (B) Rim-like membranous staining pattern of the nuclear membrane. Granular cytoplasmic staining is due to the presence of antimitochondrial antibodies. Original magnification x 40.
]. However, although a distinct increase of IgG characterizes most AIH patients, about 10–15% of chronic and up to 39% of the acute AIH cases may have indeed normal IgG at baseline [
]. It should be emphasized however, that the “normal” range of IgG in routine laboratories is wide, as it is not practical to establish the “real normal ranges” of the respective population where an index subject with AIH is living. As a result, physicians may mistakenly disregard AIH from the differential diagnosis leading to delay of diagnosis with potential catastrophic consequences for the patients. Therefore, additional testing for autoantibodies is advised even in cases with “normal” IgG levels and if positive, liver biopsy should be performed to confirm or exclude AIH.
It should be noted that even in patients with “normal” IgG, alterations of IgG could be used to monitor response to treatment, as a recent study has shown a considerable fall of IgG after immunosuppressive therapy, and a decline of IgG, sometimes below the lower limit of normal, in almost all AIH patients with “normal” IgG at diagnosis [
My patient has high titre ANA and anti-dsDNA antibodies but also moderate increase of transaminases for a long time with negative investigation for other liver disorders including viral hepatitis markers. Should I consider systemic lupus erythematosus (SLE) as the underlying diagnosis?
It should be emphasized that seropositivity for ANA and anti-dsDNA antibodies should not lead the physicians to the “superficial diagnosis” of SLE if other criteria for its diagnosis are missing, because anti-dsDNA antibodies can also be detected in about 30% of AIH patients and up to 60% of patients with AIH/PBC variant [
]. Anti-dsDNA antibodies are usually detected by ELISAs or IIF on Crithidia luciliae substrate which incorporates high quantities of dsDNA and therefore, it permits a simple and easy way to detect them with higher specificity than the molecular-based ELISAs. As many AIH patients suffer from polyarthralgia syndrome without arthritis usually involving the small joints for years, it is reasonable for physicians to consider the diagnosis of SLE instead of AIH in an index patient with ANA and anti-dsDNA positivity (Table 2). However, involvement of the liver is not normally part of the clinical spectrum of SLE. Indeed, coincidence of viral hepatitis, non-alcoholic liver disease commonly induced by corticosteroids and DILI because of SLE-related therapies are the most frequent causes of deranged liver enzymes in patients with SLE [
The key point for the suspicion of AIH in such cases is if the patients have abnormal transaminases along with high IgG serum levels and SMA with F-actin reactivity which direct towards AIH rather than SLE diagnosis. In this regard, liver biopsy will distinguish AIH from SLE as in the latter only non-specific findings are present [
]. From the clinical perspective, this misdiagnosis may have catastrophic consequences for the patients as we have seen a fair number of such cases who at initial diagnosis of AIH presented with advanced disease needing rescue therapies because of delayed diagnosis [
], have been occasionally found in patients with other liver diseases including AIH (Table 2). Indeed, AMA detection has been reported in about 5–10% (up to 35% in some Japanese studies) of typical cases of AIH without any evidence of cholestatic disease. The clinical significance of this finding is currently enigmatic. Up to the present, most studies agree that AMA presence in patients with AIH is not corresponded to a specific subgroup of AIH patients requiring different therapeutic options or to a rapid development of PBC characteristics during follow-up [
Patients with autoimmune hepatitis who have antimitochondrial antibodies need long-term follow-up to detect late development of primary biliary cirrhosis.
]. The limitations of short follow-up, infrequent sequential histologic examination and the small numbers of patients included in these studies may have led to the abovementioned inconclusive results. From the clinical point of view, clinicians should be aware of this phenomenon and manage their AIH patients with AMA reactivity as in those without if cholestatic indices are not present both at the biochemical and histological level.
4. Concluding remarks and future perspectives
AIH diagnosis is still challenging for physicians, as there is not a single test to diagnose or exclude the disease while its presentation is highly heterogeneous both at clinical and the serological level. Therefore, AIH should be absolutely considered in the differential diagnosis of all subjects with acute or chronic hepatitis of any severity irrespective of sex, age, or ethnicity. The precise guidelines for autoantibodies investigation [
] all point directly to facilitate prompt recognition of the disease, which is of utmost importance to stop the disease progression and achieve favourable outcomes after treatment initiation. In this regard, physicians should be aware for the significance of the autoantibodies testing they order and should also be familiar with the interpretation of the results.
It is clear that for a definite diagnosis of AIH, new biomarker(s) with both higher sensitivity and specificity than conventional autoantibodies are needed. Metabolomics and proteomics could be good candidates to identify in the future such specific molecules in AIH [
] discovered the presence of IgG antibodies with high capacity to bind to several human and non-human proteins by using a protein microarray. This polyreactive IgG (pIgG) showed greater specificity for the diagnosis of AIH than standard ANA and SMA tests, and a significantly higher sensitivity than anti-LKM and anti-SLA/LP [
Patients with autoimmune hepatitis who have antimitochondrial antibodies need long-term follow-up to detect late development of primary biliary cirrhosis.
Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the american association for the study of liver diseases.
Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.
Meta-analysis: diagnostic accuracy of antinuclear antibodies, smooth muscle antibodies and antibodies to a soluble liver antigen/liver pancreas in autoimmune hepatitis.
Anti-neutrophil cytoplasmic antibodies in patients with chronic liver diseases: prevalence, antigen specificity and predictive value for diagnosis of autoimmune liver disease. Swedish Internal Medicine Liver Club (SILK).
Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques.
Prompt initiation of high-dose i.v. corticosteroids seem to prevent progression to liver failure in patients with original acute severe autoimmune hepatitis.
European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis.
Autoantibody status and histological variables influence biochemical response to treatment and long-term outcomes in Japanese patients with primary biliary cirrhosis.
Patients with autoimmune hepatitis who have antimitochondrial antibodies need long-term follow-up to detect late development of primary biliary cirrhosis.
00403-4&id=gr1.jpg){kind=link}
00403-4&id=gr2.jpg){kind=link}
00403-4&id=gr3.jpg){kind=link}
00403-4&id=gr4.jpg){kind=link}
00403-4&id=gr5.jpg){kind=link}
00403-4&id=gr6.jpg){kind=link}