Correspondence

Antineutrophil cytoplasmic antibodies in autoimmune hepatitis

Serology is key to the diagnosis of autoimmune hepatitis (AIH). Clinicians need to be aware of which tests to request, how to interpret the laboratory reports, and be familiar with the laboratory methodology. If correctly tested, > 95% of AIH patients show some serological reactivity. Indirect immunofluorescence on triple rodent tissue is recommended as first screening step, since it allows the detection of all liver-relevant autoantibodies, except for anti-soluble liver antigen (SLA) antibody, which needs to be detected by molecular based assays. The threshold of immunofluorescence positivity is a titer equal or exceeding 1/40, but for patients younger than 18 years even lower titers are clinically significant. Anti-nuclear antibody (ANA) and/or anti-smooth muscle (SMA) antibody characterize type 1 AIH. ANA in AIH typically shows a homogeneous staining pattern on HEp2 cells, without any specific target antigen. Anti-SMA displays different staining patterns on indirect immunofluorescence: the vascular/glomerular (VG) and the vascular/glomerular/tubular (VGT) patterns are considered specific for AIH, whilst the V pattern can be found in a variety of diseases. Type 2 AIH, which is rare and affects mostly children/adolescents, is characterized by anti-liver kidney microsomal 1 and/or anti-liver cytosol 1 antibodies. The presence of anti-neutrophil cytoplasmic antibody (ANCA), particularly atypical p-ANCA (pANNA), points to the diagnosis of AIH, especially in absence of other autoantibodies. Since it is associated with sclerosing cholangitis and inflammatory bowel disease, these conditions have to be ruled out. The only antibody specific for AIH is anti-SLA, which is associated with a more severe disease course.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD), a general term used to describe diseases that cause chronic inflammation of the intestine. Although there are many theories concerning the etiology of CD and UC, none have been proven. Since many symptoms of CD and UC are similar, diagnosis is often difficult and relies on detailed clinical evaluation, the results of endoscopic, histologic and radiographic examination, and the exclusion of other disorders. Diagnosis of IBD in pediatric patients can often be particularly difficult because of nonspecific symptoms early in the disease. While an accurate differential diagnosis is possible with most patients, 10–15% of patients have an indeterminate form with features common to both CD and UC. With time, many of these patients are eventually classified as either CD or UC. The development of accurate, standardized, reproducible and non-invasive serological tests could provide clinicians with a valuable tool to assist in the assessment and diagnosis of IBD patients. Furthermore, accurate serological tests may assist clinicians make earlier diagnoses in pediatric patients. There has been a rapidly expanding interest and research into the identification and development of serological markers to assist in the diagnosis and management of patients with IBD. General markers of inflammation provide some information, but are not disease-specific. Autoantibodies to various antigens have been identified, but most have not proven to have adequate sensitivity, specificity or availability to be useful in the clinical laboratory. Two exceptions are ANCA (anti-neutrophil cytoplasmic antibody and ASCA (anti-Saccharomyces cerevisiae antibodies). The first part of this article will present a general overview of IBD, followed by a review of some of the markers that have been evaluated for IBD. The second part will focus specifically on the development and current experiences with ASCA assays in the laboratory.

Autoimmune hepatitis (AIH) comprises a relatively diverse group of liver diseases associated with autoantibody formation (Fig. 1) which are thought to occur as a result of an uncontrolled, self-directed inflammatory attack on hepatocytes or bile ducts. The immune injury commonly leads to rapidly progressive liver disease, progressive fibrosis, and ultimately cirrhosis. The cause of autoimmune liver disease is not known, but factors that influence the development of clinical disease include genetic predisposition, prior liver injury caused by viruses, environmental exposure to toxins, and, possibly, ongoing infectious factors, including viruses.

The incidence and prevalence of the forms of autoimmune liver diseases remain to be defined worldwide, although information is available for distinct ethnic populations.²⁷ The point prevalence of autoimmune liver disease ranges from 13 to 20/100,000 population and the incidence from 1 to 2/100,000 in a Norwegian population.³⁵

Improving the understanding of autoimmune liver disease will allow physicians and other medical practitioners to make decisions about the proper type of therapy to prevent progressive liver disease and may ultimately eliminate death from liver failure and the need for liver transplantation. Other focuses of therapy include improving quality of life preventing primary septic complications caused by biliary strictures (in primary sclerosing cholangitis) and infectious complications of immune suppression.

Serologic assays, biochemical tests, histologic criteria, and imaging tests have provided the basis for the diagnosis of autoimmune liver disease. Recent advances in serologic testing, genetic studies, and immunohistochemistry may provide more rapid and accurate diagnosis of patients with autoimmune liver disease and may also guide the cause of immunosuppressive therapy.

Infectious causes of autoimmune liver disease have been proposed, with prior viral hepatitis A, B, or C, cytomegalovirus (CMV), paramyxovirus, and most recently retrovirus infections suggested as possibilities.108, 111 The development of recurrent AIH, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC) in liver transplant recipients provides strong support for an infectious origin for of these disorders. If environmental agents are found to be the cause or a component of disease activity and progression, advances in antibiotic and antiviral therapies may also delay or prevent progressive liver disease.

Anti-neutrophil specific antibodies are detected in up to 88% of patients with PSC. The labelling pattern of neutrophils produced by these antibodies when examined by indirect immunofluorescence microscopy is distinct from that produced by anti-neutrophil cytoplasmic antibodies (ANCA) in vasculitic diseases. The antigen(s) recognized by anti-neutrophil antibodies in PSC is not yet known but appears to be localized to the periphery of the nucleus. Accordingly, the term peripheral anti-neutrophil nuclear antibodies (p-ANNA) is more appropriate than the frequently used p-ANCA. As the titre of p-ANNA in PSC does not correlate with disease-specific clinical parameters, they are not useful markers for the management of patients. However, the high prevalence of p-ANNA in PSC makes them a reasonable diagnostic marker if used in conjunction with other standard diagnostic tests. The role of anti-neutrophil antibodies in the pathogenesis of PSC, if any, remains to be established.

Objective: Lactoferrin, an immunoregulatory protein in mucosal secretions, is one of the target antigens to perinuclear antineutrophil cytoplasmic antibodies (P-ANCAs). Circulating lactoferrin is cleared in the liver, but little is known about the implication of lactoferrin in hepatic inflammation. To evaluate the implication of immunological response to lactoferrin, we examined antilactoferrin antibodies in autoimmune liver diseases. Methods: Fourteen patients with primary biliary cirrhosis (PBC), 14 with autoimmune hepatitis (AIH), five with autoimmune cholangitis (AIC), six with chronic hepatitis C, and five with chronic hepatitis B were studied. We evaluated autoantibodies to lactoferrin in the sera of the patients by the Western Immunoblotting method. Results: Sera of five of the 14 patients (35.7%) with PBC, four of the 14 patients (28.6%) with AIH, and five of the five patients (100%) with AIC contained autoantibodies to human lactoferrin, but none with hepatitis B or C had them. The higher prevalence of serum antibodies to human lactoferrin was shown to be higher in patients with AIC than with hepatitis B (p < 0.01), hepatitis C (p < 0.01), PBC (p < 0.05), and AIH (p < 0.05). Conclusion: Lactoferrin located in bile ducts and liver cells is one of the candidates of target antigens in autoimmune liver diseases, especially in AIC.