Scandinavian Journal of Gastroenterology
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, AntiCentromere, and Mitotic Spindle Antibodies B. U. Hansen, S. Eriksson & S. Lindgren To cite this article: B. U. Hansen, S. Eriksson & S. Lindgren (1991) High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, Anti-Centromere, and Mitotic Spindle Antibodies, Scandinavian Journal of Gastroenterology, 26:7, 707-713, DOI: 10.3109/00365529108998588 To link to this article: http://dx.doi.org/10.3109/00365529108998588
Published online: 08 Jul 2009.
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Date: 30 April 2016, At: 18:06
High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, Anti-Centromere, and Mitotic Spindle Antibodies B. U. HANSEN, S. ERIKSSON & S. LINDGREN Sections of Rheumatology and Gastroenterology-Hepatology,Dept. of Medicine Malmo, University of Lund, Malmo General Hospital, Malmo, Sweden
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Hansen BU, Eriksson S, Lindgren S. High prevalence of autoimmune liver disease in patients with multiple nuclear dot, anti-centromere, and mitotic spindle antibodies. Scand J Gastroenterol 1991, 26, 707-713 We used HEp-2 cells to compare the Occurrence of multiple nuclear dot (MND), anti-centromere (ACA), and mitotic spindle antibodies (MSA) in patients with primary biliary cirrhosis (PBC) ( n = 32) and primary Sjogren’s syndrome (n = 50). The predictive value of these antibodies for autoimmune liver disease was evaluated among patients with chronic liver or inflammatory connective tissue diseases. MND were found in 6%, ACA in lo%, and MSA in 6% of PBC sera. Among patients with primary Sjogren’s syndrome, ACA were found in one, whereas another had both MND and MSA. MND were also detected in 1 of 25 sera from patients with other chronic inflammatory connective tissue diseases. Reexamination of these three patients showed evidence of PBC in two. In a blinded study of sera from 356 patients with chronic liver diseases, MND were detected in 10 (3%), ACA in 2 (0.9%), and MSA in 4 (1.2%).All patients with MND or ACA and two of four with MSA had PBC or autoimmune chronic active hepatitis, particularly of the cholestatic type. In four of these patients the liver disease had not previously been classified. We conclude that these antibodies have low sensitivity but high predictive value for autoimmune, cholestatic liver disease. Key words: Anti-nuclear antibodies; autoimmune liver disease; chronic liver disease; connective tissue disease; primary biliary cirrhosis; primary Sjogren’s syndrome Stefan Lindgren, M . D., Dept. of Medicine, Malmci General Hospital, S-214 01 Malmo, Sweden
Patients with autoimmune liver diseases and chronic inflammatory connective tissue disorders have an increased prevalence of anti-nuclear antibodies (ANA) (1). We have previously reported a high prevalence of focal sialadenitis and moderately increased levels of serum anti-SS-B/La antibodies in patients with primary biliary cirrhosis (PBC) (2). Features of several autoimmune diseases are often found in patients with autoimmune disorders, although clinical manifestations are limited (3). The frequency of these overlapping forms is largely unknown. Cholestatic autoimmune chronic active hepatitis (CAH) probably represents a form overlapping with PBC (4).
The availability of HEp-2 cells has markedly facilitated detection of several ANA patterns by indirect immunofluorescence (5). Of particular interest are three readily recognizable antibody patterns: anti-centromere antibodies (ACA), multiple nuclear dot antibodies (MND), and mitotic spindle apparatus antibodies (MSA). The ACA give a speckled pattern of the nucleus in metaphase (Fig. la) (6). The ACA are primarily considered markers of the CREST (calcinosis, Raynaud, esophageal dysfunction, sclerodactyly , telangiectasia) syndrome (7). MND are characteristicaHy seen as 8-12 rather large spots in the nucleus in the interphase cell (Fig. lb). The chemical nature of the antigenic nuclear bodies
Downloaded by [McMaster University] at 18:06 30 April 2016
708
B. U.Hansen et a/.
Downloaded by [McMaster University] at 18:06 30 April 2016
ANA in Autoimmune Liver Disease
remains unknown (8). Both ACA and MND have been found in association with PBC (9,lO). The MSA recognize the centrisome in mitotic cells (Fig. lc), and the MSA pattern is due to antibodies against the nuclear mitotic antigen NuMA, which is of high molecular weight but otherwise uncharacterized (11). The purpose of this investigation was primarily to evaluate the occurrence of these three antibodies in patients with well-defined PBC and primary Sjogren’s syndrome. Secondly, we determined their sensitivity and predictive value as markers for autoimmune liver disease on blinded analysis of sera from patients with chronic liver or inflammatory connective tissue disorders.
PATIENTS AND METHODS Patients Sera were obtained from 32 patients with PBC (12) (mean age, 59 years; range, 46-80; male to female ratio, 3:29), 50 patients with objectively verified keratoconjunctivitis sicca and xerostomia in accordance with the Copenhagen criteria for primary Sjogren’s syndrome (13) (mean age, 60 years; range, 21-81; male to female ratio, 1:24), 25 patients with various chronic inflammatory connective tissue disorders (mean age, 52 years; range, 20-75; male to female ratio, 2:23), 346 patients with chronic liver diseases (mean age, 57 years; range, 19-80; male to female ratio, 3 : l), and 20 patients with viral hepatitis (5, acute hepatitis B; 10, chronic hepatitis B; 5, acute hepatitis A). Sera from 25 randomly selected patients hospitalized at the Dept. of Medicine were also included (mean age, 75 years; range, 62-83; male to female ratio, 1:1). The patients with PBC and primary Sjogren’s Fig. 1. Indirect immunofluorescence antinuclear antiin Sera bodv test with human eDithelial ( H E D - ~cells ) from patients with autoimmune liver diseases. la. The nuclear staining represents multiple nuclear dot antibodies (MND) (arrow), and the cytoplasmic staining represents anti-mitochondfial antibodies (AMA) (airowhead). lb. The nuclear staining represents anticentromere antibodies (ACA) (arrow), and the CYtoplasmic staining represents AMA. lc. Anti-mitotic spindle apparatus antibodies indicated by arrow. Diameter of cell nuclei = 12 pm. \--
1
I
709
syndrome represented most of the patients with these diseases diagnosed and studied at the Dept. of Medicine (12,13). Sera from 21 of the patients with PBC were also included in the group with chronic liver diseases. The chronic connective tissue disorders included rheumatoid arthritis (n = 10) in accordance with the American Rheumatism Association (ARA) classification criteria (14), systemic lupus erythematosus (SLE) (n = 3) in accordance with the A R A criteria for classification (15), mixed connective tissue disease (n = 3) (16), progressive systemic sclerosis (n = 3), and unclassified connective tissue diseases (n = 6) in which two or more organ systems (such as joints, muscles, skin) were involved. The sera from patients with chronic liver diseases represented the past 5 years in a large collection of frozen serum samples obtained prospectively during diagnostic study or follow-up of patients with liver diseases for 10 years. The diagnosis was based on physical examination; biochemical screening, including bilirubin, aminotransferases, gamma-glutamyl transpeptidase, alkaline phosphatases, iron, total iron-binding capacity, ferritin, al-antitrypsin, ceruloplasmin, immunoglobulins (IgG, IgA, IgM), and a-fetoprotein; viral studies (HBsAg); and detection of autoantibodies (ANA, antimitochondrial antibodies (AMA), smooth-muscle antibodies) by indirect immunofluorescence. In most cases ultrasonographic examination of the liver was performed, and in selected cases endoscopic retrograde cholangiopancreatography (ERCP) . A percutaneous liver biopsy was performed in 96 patients. Alcohol-related liver disease was predominant (n = 148). The other sera were collected from patients with metabolic liver disease (a,-antitrypsin deficiency, hemochromatosis, Wilson’s disease, n = 15), autoimmune CAH (n = 25), PBC (n = 21), non-A, non-B hepatitis (n = 121, hepatitis B (n = 8), primary sclerosing cholangitis (n = 6 ) . svstemic diseases In = 23). non-alcoholic steathosis (n = 38), drug-related liver disease (non-steroid anti-inflammatory drugs, sedatives-hypnotics, anti-depressants, phenotiazines, n = 24), cryptogenic cirrhosis (n = lo), and liver disease (n = 16). The patients classified as having autoimmune v
,
I
,
2
I~
710
B. U.Hansen et al.
Downloaded by [McMaster University] at 18:06 30 April 2016
CAH all had elevated plasma levels of IgG, antismooth muscle antibodies, and/or ANA (17). The patients with primary sclerosing cholangitis had characteristic extrahepatic bile duct lesions demonstrated by ERCP and a liver biopsy compatible with the diagnosis. Retrospectively, 100 of 346 sera were tested for anti-hepatitis C antibodies by enzyme-linked immunosorbent assay (ELISA) (Ortho). This included sera from the 12 patients classified as having non-A, non-B hepatitis.
rescence pattern with regard to MND, ACA, and MSA by the two independent observers were concordant; that is, both classified the same 28 sera as positive for these antibodies. In all cases of MND, ACA, or MSA the end-point titer was >1/1000. The nuclear staining patterns produced by sera from patients with PBC, primary Sjogren’s syndrome, or chronic inflammatory connective tissue diseases are presented in Table I. Most patients with primary Sjogren’s syndrome and positive ANA ( n = 42) had a fine speckled pattern ( n = 25). Reexamination of the single patient with Sjogren’s syndrome and MND showed a chronic Immunofluorescence studies The presence of ANA was detected by indirect cholestatic liver disease with high-titer AMA. immunofluorescence. Serum diluted 1:40 was This patient also had MSA antibodies. The single incubated for 30 rnin on HEp-2 cells (Kallestad, patient with primary Sjogren’s syndrome and Austin, Texas) at 24°C in a humid chamber. After ACA had typical Raynaud phenomena but no being washed for 15 rnin in phosphate-buffered other manifestations of the CREST syndrome and saline (PBS), fluorescein isothiocyanate (FITC)- no clinical or laboratory signs of liver disease. labeled rabbit anti-human IgG (DAKO, Cop- One patient with unclassified chronic connective enhagen, Denmark) diluted 1:25 was added and tissue disease had MND. Further examination incubated for 30 min in a humid chamber. After showed a chronic cholestatic liver disease with a final washing for 15 min the slides were exam- elevated plasma IgM levels and objectively veriined in a BH-2 Olympus fluorescence microscope fied keratoconjunctivitis sicca without verifiable 0 objective. An ANA-positive xerostomia. AMA was negative, but the liver with the ~ 4 dry and a negative control were included on each slide biopsy was compatible with PBC. Homogeneous (18). For all positive tests, the procedure was or speckled nuclear fluorescence was produced by repeated, and a titer was determined to ascertain sera from 3 of 25 elderly hospitalized patients. the highest dilution to give a positive result. All None had ACA, MND, or MSA. The results from blindly studied sera from tests were run without knowledge of the clinical patients with various chronic liver diseases are diagnosis, and the pattern of fluorescence was presented in Table 11. A total of 17 patients with evaluated by two independent observers. MND, ACA, or MSA were found. Of these, eight had PBC (47%) and seven had autoimmune CAH RESULTS (41%) with smooth-muscle antibodies and high The results of evaluation of the immunofluo- plasma IgG levels. Four of the eight patients with Table I. Analysis of antinuclear antibodies (ANA) in sera from patients with primary biliary cirrhosis (PBC), primary Sjogren’s syndrome (pSS), and chronic inflammatory connective tissue diseases (CTD) by indirect immunofluorescence of HEp-2 cells (Ab = antibodies) Diagnosis Staining pattern Overall prevalence of homogeneous or speckled ANA Anti-centromere Ab Multiple nuclear dot Ab
Mitotic spindle Ab
PBC, n
= 32
7 (22%) 3 (10%) 2 (6%) 2 (6%)
pSS, a = 50
CTD, n = 25
42 (85%)
13 (52%)
1(2%) 1(2%) 1(2%)
0
1(4%) 0
ANA in Autoimmune Liver Dbease
DISCUSSION Antinuclear antibodies as detected by indirect immunofluorescence are widely used serologic screening markers of connective tissue disease. Different ANA patterns show remarkable but not absolute disease associations (3). The speckled No. with antibodies nuclear pattern, for example, is due to antibodies (titer > 1/1000) against the extractable nuclear antigens Sm, RNP, Scl-70, or SS-B/La, which are associated with Antibodies n % SLE, mixed connective tissue disease, progresAnti-centromere 3 0.9 sive systemic sclerosis, and primary Sjogren’s 1 PBC, 2 syndrome, respectively. Homogeneous staining cholestatic autoimmune CAH of the nucleus indicates anti-single or -doubleMultiple nuclear dot 10 3 stranded DNA antibodies or anti-histone anti6 PBC (3 not bodies. When present in high titer, it is suggestive previously classified) of SLE or drug-induced SLE. However, for 4 autoimmune specific antibody determination other serologic CAH ( 3 methods such as precipitation, radioimmunocholestatic) Mitotic spindle 4 1.2 assay, or ELISA are needed (18). 1 PBC (not The human epidermal tissue culture cell line, previously HEp-2 cells, combines access to easily difclassified) 1 autoimmune CAH ferentiated cellular details and a high percentage of mitotic cells with the presence of many specific nuclear antigens desired in a substrate for ANA screening (18). A few ANA patterns on HEp-2 PBC were classified as having PBC after further cells are related to single, morphologically easily diagnostic evaluation including immunoglobulin identifiable nuclear a n t i g e n e t h a t is, ACA and analysis, AMA test, ultrasonographic liver exam- MSA (6,s).These patterns are not recognized ination, and liver biopsy. Five of the patients with in conventional immunofluorescence ANA tests autoimmune CAH had a pronounced biochemical using kidney or liver tissues as substrate. ACA cholestasis, but all lacked AMA. No evidence have been reported to be present in 5&95% of of autoimmune liver disease was found in two patients with the CREST syndrome (9, 19). In patients with MSA, both with malignant liver addition, ACA have been observed in approxidisease. All patients with hepatitis A or B were mately 10% of patients with PBC, of whom 60% ANA-negative. Anti-hepatitis C antibodies were were reported to have features of CREST (10). detected by ELISA in 24 of 100 sera tested. All In the present study we found a high prevalence patients classified as having non-A, non-B hepa- of PBC among patients with ACA. We could titis had anti-hepatitis C antibodies with optical not, however, correlate the occurrence of these density (OD) values > 2.0. In addition, inter- antibodies with the classical clinical picture of mediate O D values ( l . b l . 5 ) were detected in CREST. ACA were also seen in patients with two patients with PBC, two with autoimmune autoimmune CAH. This lack of specificity of CAH, and eight patients with cryptogenic or ACA as a marker for the CREST syndrome is unclassified liver disease. One of the hepatitis C- supported by other recent observations (20). positive PBC patients had MND, but the other In our study MND had a 100% predictive value 23 sera were negative for ACA, MND, and MSA. for autoimmune liver disease, even when detected The overall sensitivity of these three antibodies in patients considered to have primary connective for detection of autoimmune liver disease was low tissue disorders. This extends previous obser(
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, AntiCentromere, and Mitotic Spindle Antibodies B. U. Hansen, S. Eriksson & S. Lindgren To cite this article: B. U. Hansen, S. Eriksson & S. Lindgren (1991) High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, Anti-Centromere, and Mitotic Spindle Antibodies, Scandinavian Journal of Gastroenterology, 26:7, 707-713, DOI: 10.3109/00365529108998588 To link to this article: http://dx.doi.org/10.3109/00365529108998588
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 13
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [McMaster University]
Date: 30 April 2016, At: 18:06
High Prevalence of Autoimmune Liver Disease in Patients with Multiple Nuclear Dot, Anti-Centromere, and Mitotic Spindle Antibodies B. U. HANSEN, S. ERIKSSON & S. LINDGREN Sections of Rheumatology and Gastroenterology-Hepatology,Dept. of Medicine Malmo, University of Lund, Malmo General Hospital, Malmo, Sweden
Downloaded by [McMaster University] at 18:06 30 April 2016
Hansen BU, Eriksson S, Lindgren S. High prevalence of autoimmune liver disease in patients with multiple nuclear dot, anti-centromere, and mitotic spindle antibodies. Scand J Gastroenterol 1991, 26, 707-713 We used HEp-2 cells to compare the Occurrence of multiple nuclear dot (MND), anti-centromere (ACA), and mitotic spindle antibodies (MSA) in patients with primary biliary cirrhosis (PBC) ( n = 32) and primary Sjogren’s syndrome (n = 50). The predictive value of these antibodies for autoimmune liver disease was evaluated among patients with chronic liver or inflammatory connective tissue diseases. MND were found in 6%, ACA in lo%, and MSA in 6% of PBC sera. Among patients with primary Sjogren’s syndrome, ACA were found in one, whereas another had both MND and MSA. MND were also detected in 1 of 25 sera from patients with other chronic inflammatory connective tissue diseases. Reexamination of these three patients showed evidence of PBC in two. In a blinded study of sera from 356 patients with chronic liver diseases, MND were detected in 10 (3%), ACA in 2 (0.9%), and MSA in 4 (1.2%).All patients with MND or ACA and two of four with MSA had PBC or autoimmune chronic active hepatitis, particularly of the cholestatic type. In four of these patients the liver disease had not previously been classified. We conclude that these antibodies have low sensitivity but high predictive value for autoimmune, cholestatic liver disease. Key words: Anti-nuclear antibodies; autoimmune liver disease; chronic liver disease; connective tissue disease; primary biliary cirrhosis; primary Sjogren’s syndrome Stefan Lindgren, M . D., Dept. of Medicine, Malmci General Hospital, S-214 01 Malmo, Sweden
Patients with autoimmune liver diseases and chronic inflammatory connective tissue disorders have an increased prevalence of anti-nuclear antibodies (ANA) (1). We have previously reported a high prevalence of focal sialadenitis and moderately increased levels of serum anti-SS-B/La antibodies in patients with primary biliary cirrhosis (PBC) (2). Features of several autoimmune diseases are often found in patients with autoimmune disorders, although clinical manifestations are limited (3). The frequency of these overlapping forms is largely unknown. Cholestatic autoimmune chronic active hepatitis (CAH) probably represents a form overlapping with PBC (4).
The availability of HEp-2 cells has markedly facilitated detection of several ANA patterns by indirect immunofluorescence (5). Of particular interest are three readily recognizable antibody patterns: anti-centromere antibodies (ACA), multiple nuclear dot antibodies (MND), and mitotic spindle apparatus antibodies (MSA). The ACA give a speckled pattern of the nucleus in metaphase (Fig. la) (6). The ACA are primarily considered markers of the CREST (calcinosis, Raynaud, esophageal dysfunction, sclerodactyly , telangiectasia) syndrome (7). MND are characteristicaHy seen as 8-12 rather large spots in the nucleus in the interphase cell (Fig. lb). The chemical nature of the antigenic nuclear bodies
Downloaded by [McMaster University] at 18:06 30 April 2016
708
B. U.Hansen et a/.
Downloaded by [McMaster University] at 18:06 30 April 2016
ANA in Autoimmune Liver Disease
remains unknown (8). Both ACA and MND have been found in association with PBC (9,lO). The MSA recognize the centrisome in mitotic cells (Fig. lc), and the MSA pattern is due to antibodies against the nuclear mitotic antigen NuMA, which is of high molecular weight but otherwise uncharacterized (11). The purpose of this investigation was primarily to evaluate the occurrence of these three antibodies in patients with well-defined PBC and primary Sjogren’s syndrome. Secondly, we determined their sensitivity and predictive value as markers for autoimmune liver disease on blinded analysis of sera from patients with chronic liver or inflammatory connective tissue disorders.
PATIENTS AND METHODS Patients Sera were obtained from 32 patients with PBC (12) (mean age, 59 years; range, 46-80; male to female ratio, 3:29), 50 patients with objectively verified keratoconjunctivitis sicca and xerostomia in accordance with the Copenhagen criteria for primary Sjogren’s syndrome (13) (mean age, 60 years; range, 21-81; male to female ratio, 1:24), 25 patients with various chronic inflammatory connective tissue disorders (mean age, 52 years; range, 20-75; male to female ratio, 2:23), 346 patients with chronic liver diseases (mean age, 57 years; range, 19-80; male to female ratio, 3 : l), and 20 patients with viral hepatitis (5, acute hepatitis B; 10, chronic hepatitis B; 5, acute hepatitis A). Sera from 25 randomly selected patients hospitalized at the Dept. of Medicine were also included (mean age, 75 years; range, 62-83; male to female ratio, 1:1). The patients with PBC and primary Sjogren’s Fig. 1. Indirect immunofluorescence antinuclear antiin Sera bodv test with human eDithelial ( H E D - ~cells ) from patients with autoimmune liver diseases. la. The nuclear staining represents multiple nuclear dot antibodies (MND) (arrow), and the cytoplasmic staining represents anti-mitochondfial antibodies (AMA) (airowhead). lb. The nuclear staining represents anticentromere antibodies (ACA) (arrow), and the CYtoplasmic staining represents AMA. lc. Anti-mitotic spindle apparatus antibodies indicated by arrow. Diameter of cell nuclei = 12 pm. \--
1
I
709
syndrome represented most of the patients with these diseases diagnosed and studied at the Dept. of Medicine (12,13). Sera from 21 of the patients with PBC were also included in the group with chronic liver diseases. The chronic connective tissue disorders included rheumatoid arthritis (n = 10) in accordance with the American Rheumatism Association (ARA) classification criteria (14), systemic lupus erythematosus (SLE) (n = 3) in accordance with the A R A criteria for classification (15), mixed connective tissue disease (n = 3) (16), progressive systemic sclerosis (n = 3), and unclassified connective tissue diseases (n = 6) in which two or more organ systems (such as joints, muscles, skin) were involved. The sera from patients with chronic liver diseases represented the past 5 years in a large collection of frozen serum samples obtained prospectively during diagnostic study or follow-up of patients with liver diseases for 10 years. The diagnosis was based on physical examination; biochemical screening, including bilirubin, aminotransferases, gamma-glutamyl transpeptidase, alkaline phosphatases, iron, total iron-binding capacity, ferritin, al-antitrypsin, ceruloplasmin, immunoglobulins (IgG, IgA, IgM), and a-fetoprotein; viral studies (HBsAg); and detection of autoantibodies (ANA, antimitochondrial antibodies (AMA), smooth-muscle antibodies) by indirect immunofluorescence. In most cases ultrasonographic examination of the liver was performed, and in selected cases endoscopic retrograde cholangiopancreatography (ERCP) . A percutaneous liver biopsy was performed in 96 patients. Alcohol-related liver disease was predominant (n = 148). The other sera were collected from patients with metabolic liver disease (a,-antitrypsin deficiency, hemochromatosis, Wilson’s disease, n = 15), autoimmune CAH (n = 25), PBC (n = 21), non-A, non-B hepatitis (n = 121, hepatitis B (n = 8), primary sclerosing cholangitis (n = 6 ) . svstemic diseases In = 23). non-alcoholic steathosis (n = 38), drug-related liver disease (non-steroid anti-inflammatory drugs, sedatives-hypnotics, anti-depressants, phenotiazines, n = 24), cryptogenic cirrhosis (n = lo), and liver disease (n = 16). The patients classified as having autoimmune v
,
I
,
2
I~
710
B. U.Hansen et al.
Downloaded by [McMaster University] at 18:06 30 April 2016
CAH all had elevated plasma levels of IgG, antismooth muscle antibodies, and/or ANA (17). The patients with primary sclerosing cholangitis had characteristic extrahepatic bile duct lesions demonstrated by ERCP and a liver biopsy compatible with the diagnosis. Retrospectively, 100 of 346 sera were tested for anti-hepatitis C antibodies by enzyme-linked immunosorbent assay (ELISA) (Ortho). This included sera from the 12 patients classified as having non-A, non-B hepatitis.
rescence pattern with regard to MND, ACA, and MSA by the two independent observers were concordant; that is, both classified the same 28 sera as positive for these antibodies. In all cases of MND, ACA, or MSA the end-point titer was >1/1000. The nuclear staining patterns produced by sera from patients with PBC, primary Sjogren’s syndrome, or chronic inflammatory connective tissue diseases are presented in Table I. Most patients with primary Sjogren’s syndrome and positive ANA ( n = 42) had a fine speckled pattern ( n = 25). Reexamination of the single patient with Sjogren’s syndrome and MND showed a chronic Immunofluorescence studies The presence of ANA was detected by indirect cholestatic liver disease with high-titer AMA. immunofluorescence. Serum diluted 1:40 was This patient also had MSA antibodies. The single incubated for 30 rnin on HEp-2 cells (Kallestad, patient with primary Sjogren’s syndrome and Austin, Texas) at 24°C in a humid chamber. After ACA had typical Raynaud phenomena but no being washed for 15 rnin in phosphate-buffered other manifestations of the CREST syndrome and saline (PBS), fluorescein isothiocyanate (FITC)- no clinical or laboratory signs of liver disease. labeled rabbit anti-human IgG (DAKO, Cop- One patient with unclassified chronic connective enhagen, Denmark) diluted 1:25 was added and tissue disease had MND. Further examination incubated for 30 min in a humid chamber. After showed a chronic cholestatic liver disease with a final washing for 15 min the slides were exam- elevated plasma IgM levels and objectively veriined in a BH-2 Olympus fluorescence microscope fied keratoconjunctivitis sicca without verifiable 0 objective. An ANA-positive xerostomia. AMA was negative, but the liver with the ~ 4 dry and a negative control were included on each slide biopsy was compatible with PBC. Homogeneous (18). For all positive tests, the procedure was or speckled nuclear fluorescence was produced by repeated, and a titer was determined to ascertain sera from 3 of 25 elderly hospitalized patients. the highest dilution to give a positive result. All None had ACA, MND, or MSA. The results from blindly studied sera from tests were run without knowledge of the clinical patients with various chronic liver diseases are diagnosis, and the pattern of fluorescence was presented in Table 11. A total of 17 patients with evaluated by two independent observers. MND, ACA, or MSA were found. Of these, eight had PBC (47%) and seven had autoimmune CAH RESULTS (41%) with smooth-muscle antibodies and high The results of evaluation of the immunofluo- plasma IgG levels. Four of the eight patients with Table I. Analysis of antinuclear antibodies (ANA) in sera from patients with primary biliary cirrhosis (PBC), primary Sjogren’s syndrome (pSS), and chronic inflammatory connective tissue diseases (CTD) by indirect immunofluorescence of HEp-2 cells (Ab = antibodies) Diagnosis Staining pattern Overall prevalence of homogeneous or speckled ANA Anti-centromere Ab Multiple nuclear dot Ab
Mitotic spindle Ab
PBC, n
= 32
7 (22%) 3 (10%) 2 (6%) 2 (6%)
pSS, a = 50
CTD, n = 25
42 (85%)
13 (52%)
1(2%) 1(2%) 1(2%)
0
1(4%) 0
ANA in Autoimmune Liver Dbease
DISCUSSION Antinuclear antibodies as detected by indirect immunofluorescence are widely used serologic screening markers of connective tissue disease. Different ANA patterns show remarkable but not absolute disease associations (3). The speckled No. with antibodies nuclear pattern, for example, is due to antibodies (titer > 1/1000) against the extractable nuclear antigens Sm, RNP, Scl-70, or SS-B/La, which are associated with Antibodies n % SLE, mixed connective tissue disease, progresAnti-centromere 3 0.9 sive systemic sclerosis, and primary Sjogren’s 1 PBC, 2 syndrome, respectively. Homogeneous staining cholestatic autoimmune CAH of the nucleus indicates anti-single or -doubleMultiple nuclear dot 10 3 stranded DNA antibodies or anti-histone anti6 PBC (3 not bodies. When present in high titer, it is suggestive previously classified) of SLE or drug-induced SLE. However, for 4 autoimmune specific antibody determination other serologic CAH ( 3 methods such as precipitation, radioimmunocholestatic) Mitotic spindle 4 1.2 assay, or ELISA are needed (18). 1 PBC (not The human epidermal tissue culture cell line, previously HEp-2 cells, combines access to easily difclassified) 1 autoimmune CAH ferentiated cellular details and a high percentage of mitotic cells with the presence of many specific nuclear antigens desired in a substrate for ANA screening (18). A few ANA patterns on HEp-2 PBC were classified as having PBC after further cells are related to single, morphologically easily diagnostic evaluation including immunoglobulin identifiable nuclear a n t i g e n e t h a t is, ACA and analysis, AMA test, ultrasonographic liver exam- MSA (6,s).These patterns are not recognized ination, and liver biopsy. Five of the patients with in conventional immunofluorescence ANA tests autoimmune CAH had a pronounced biochemical using kidney or liver tissues as substrate. ACA cholestasis, but all lacked AMA. No evidence have been reported to be present in 5&95% of of autoimmune liver disease was found in two patients with the CREST syndrome (9, 19). In patients with MSA, both with malignant liver addition, ACA have been observed in approxidisease. All patients with hepatitis A or B were mately 10% of patients with PBC, of whom 60% ANA-negative. Anti-hepatitis C antibodies were were reported to have features of CREST (10). detected by ELISA in 24 of 100 sera tested. All In the present study we found a high prevalence patients classified as having non-A, non-B hepa- of PBC among patients with ACA. We could titis had anti-hepatitis C antibodies with optical not, however, correlate the occurrence of these density (OD) values > 2.0. In addition, inter- antibodies with the classical clinical picture of mediate O D values ( l . b l . 5 ) were detected in CREST. ACA were also seen in patients with two patients with PBC, two with autoimmune autoimmune CAH. This lack of specificity of CAH, and eight patients with cryptogenic or ACA as a marker for the CREST syndrome is unclassified liver disease. One of the hepatitis C- supported by other recent observations (20). positive PBC patients had MND, but the other In our study MND had a 100% predictive value 23 sera were negative for ACA, MND, and MSA. for autoimmune liver disease, even when detected The overall sensitivity of these three antibodies in patients considered to have primary connective for detection of autoimmune liver disease was low tissue disorders. This extends previous obser(
