Serial Liver Biopsies Research Articles

Autoimmune cholangiopathy: Part of the spectrum of autoimmune chronic active hepatitis

We describe four patients with features overlapping those of primary biliary cirrhosis and autoimmune chronic active hepatitis. Three were female and one was male; only one was symptomatic. Serum biochemical study showed increases in alkaline phosphatase and alpha-glutamyltranspeptidase levels. Markers of hepatitis B and C viruses were absent. In all four patients, serum mitochondrial antibodies could not be detected on immunofluorescence study and serum M2 antibodies were absent. All four patients had high titers of serum antinuclear antibody of diffuse type. Serum actin antibodies were detected in all four patients. Liver biopsy specimens showed histological features of primary biliary cirrhosis, with marked cellular infiltration of the portal areas and bile duct damage. Intralobular inflammation and piecemeal necrosis were mild. Three patients were treated with prednisolone and showed rapid clinical and biochemical remission. Serial liver biopsy specimens showed reduced inflammation, but bile duct lesions persisted. These patients probably form a subgroup of autoimmune chronic active type 1 with predominant bile duct damage. The subgroup might be termed autoimmune cholangiopathy.

Evidence that hepatitis D virus needs hepatitis B virus to cause hepatocellular damage.

Serial liver biopsy specimens from 11 patients who had liver transplants after hepatitis D virus (HDV)-related end-stage liver disease developed were examined, allowing a novel opportunity to study the evolution of HDV infection in relation to hepatitis B virus (HBV) infection from the earliest stages. Hepatitis D virus antigen was detected in liver tissue in the absence of either tissue or serologic evidence of HBV recurrence within 3 months in all eight patients biopsied at that time. After serologic evidence of recurrent HBV infection in nine patients, there was a massive increase in hepatic expression of hepatitis D virus antigen and this was associated with the transient appearance of serum hepatitis D virus antigen in four patients. Coexpression of both HBV and HDV antigens in the liver was associated with the onset of lobular hepatitis, which progressed to chronic hepatitis in five patients. These findings indicate that HDV can survive and synthesize HDAg in the absence of detectable HBV, but when HBV replication increased to a detectable level, HDV replication was enhanced massively. Contrary to current thinking, the data suggest that HDV is not directly cytopathic and that HBV is an essential cofactor in the evolution of hepatocellular damage.

Hepatitis C viral infection in liver transplant recipients.

In this study we examined multiple serial liver biopsy specimens from liver transplant recipients to determine the pathological features of hepatitis C virus-induced hepatitis. Hepatitis C virus infections acquired after transplantation and previous infections that recurred in patients after transplantation were confirmed by the results of the polymerase chain reaction. Of 43 patients infected with the hepatitis C virus, 18 had a mild form of chronic hepatitis. Four patients had hepatitis that progressed to focal bridging fibrosis or cirrhosis. There were no significant clinical or pathological differences between infections acquired after transplantation and recurrent infections (as determined by polymerase chain reaction) except that acquired infections more often developed into hepatitis. Findings indicative of hepatitis C infection included portal and parenchymal mononuclear infiltrates of varying degrees, acidophilic necrosis and swollen hepatocytes. Other common findings included lymphoid aggregates, bile duct damage and fatty change. Atypical pathological conditions included extensive hepatocyte swelling or acidophilic necrosis with minimal inflammation mimicking ischemia and ductal or ductular damage and proliferation with mixed portal infiltrates mimicking rejection or obstruction. We conclude that in transplant recipients infection by the hepatitis C virus usually produces a mild disease state, but the diagnosis of hepatitis can be difficult to make because indicators of hepatitis may mimic those of rejection, ischemia, obstruction or other hepatic infections. Serial biopsy specimens with persistent pathology and polymerase chain reaction may be necessary to define the presence of a hepatitis C virus lesion.

Cytomegalovirus infection persists in the liver graft in the vanishing bile duct syndrome.

Cytomegalovirus infection is one factor implicated in the cause of the vanishing bile duct syndrome complicating liver transplantation. To further investigate the role of cytomegalovirus in this syndrome, we studied serial liver biopsy material by in situ hybridization for cytomegalovirus DNA using a highly sensitive technique that allows the localization of viral replication. Cytomegalovirus DNA was identified in hepatocytes in 10 of 12 patients with the vanishing bile duct syndrome, 1 of whom had no serological evidence of cytomegalovirus infection. It was also present in all 18 patients with uncomplicated cytomegalovirus infection but was not identified in any of 10 subjects with transplants who had neither complication. Nine of the patients in this series underwent a diagnostic liver biopsy at 1 wk and subsequently had cytomegalovirus infection develop; cytomegalovirus DNA was identified in liver tissue of all nine patients, indicating that cytomegalovirus replication commences at an early stage. In those with uncomplicated cytomegalovirus, infection occurred earlier (p less than 0.05) but was eliminated more quickly (p less than 0.0005), and the number of infected hepatocytes was greater (p less than 0.05) when compared with those with the vanishing bile duct syndrome; in these, cytomegalovirus DNA was detectable until death or retransplantation. Cytomegalovirus DNA was never identified in either biliary or endothelial tissue. These data indicate that the vanishing bile duct syndrome is associated with persistent cytomegalovirus replication within hepatocytes.

Combination therapy with ursodeoxycholic acid and colchicine for primary biliary cirrhosis.

Twelve patients with primary biliary cirrhosis (PBC), stages I to III, received long-term therapy with a combination of 600 mg ursodeoxycholic acid (UDCA) and 1 mg colchicine given daily for more than 2 years. Drug toxicity was mild; one patient experienced diarrhoea that was probably due to colchicine. Serum levels of bilirubin, alkaline phosphatase (ALPase), gamma-glutamyl transpeptidase and alanine aminotransferase decreased by more than 50% of the initial values. Serum albumin and cholesterol levels also improved, but immunoglobulins and anti-mitochondrial antibody titre did not change. Histologic features in the eight patients who received serial liver biopsies before and 2 years after the beginning of treatment were evaluated. Piecemeal necrosis and portal inflammation were improved, but there was no change in portal fibrosis. Patients were divided into two groups; the first received both drugs from the outset, and the second group were started on UDCA for 3 months followed by the addition of colchicine. After 3 months, the improvement in serum bilirubin and ALPase in the first group was greater than in the second. However, in the second group, the ALPase levels had decreased significantly when measured at 6 and 9 months after the treatment compared with the levels at 3 months. These findings suggest that UDCA and colchicine may have a synergistic effect. This combination therapy appears to be safe and effective, both clinically and histologically, for treating PBC.

Course of hepatitis B and D virus infection in auxiliary liver grafts in hepatitis B-positive patients: A light-microscopic and immunohistochemical study

Four patients who received an auxiliary partial liver graft for decompensated liver cirrhosis due to hepatitis B (HBV), associated in two cases with hepatitis D virus (HDV) superinfection, were studied. The sequential appearance of hepatitis B and D antigens in the grafts was investigated in serial liver biopsies by immuno-histochemical methods and compared with the viral antigenic profiles of the host livers. The histological changes in the liver grafts were studied in relation to the viral expression patterns. One week after transplantation, expression of HBsAg was already apparent in two grafts. HBcAg was found in the graft of the only patient with HBcAg in the host liver. HDAg was expressed in the grafts of both patients with HDV superinfection; in one of these cases HDAg was present without HBsAg. At 3 months, viral antigen expression was maximal. Expression of HBsAg and HBcAg in the grafts of the two HDV-positive patients was, however, less extensive than in the two HBV-positive patients. All patients developed a mild lobular hepatitis, histologically demonstrated between the 47th and 107th posttransplantation day. In the two HBV-positive, HDV-negative patients, cirrhotic tranformation of the graft occurred within 1 year. In the HDV-positive patients only a mild chronic active hepatitis with slight or moderate fibrosis was observed after 1 year. We conclude that recurrence of HBV and HDV infection in auxiliary liver grafts is demonstrable within 1–3 weeks. HBV infection in liver grafts may be a rapidly progressive disease. Coinfection with HDV does not aggravate the acute hepatitis and may even suppress the progression of chronic HBV.

Histological behaviour of chronic hepatitis in patients treated with alpha interferon

To evaluate the histological effects of alpha Interferon (IFN) therapy, serial liver biopsy specimens from 30 patients with chronic hepatitis were studied. The biopsies were examined using a scoring system. After 12 mths of IFN therapy responders were 8 out of 11 HBV infected patients, 10 out of 12 HCV infected patients and only 1 out of 7 patients with cryptogenetic hepatitis. As spontaneous improvement of hepatic changes is infrequent, our data indicate that in terms of histological patterns interferon therapy is effective in chronic viral hepatitis.

Effect of platelet count on the DDAVP-induced shortening of the bleeding time in thrombocytopenic Gaucher's patients.

As part of an investigative protocol requiring serial liver biopsies in patients with Type I Gaucher's disease who were receiving enzyme replacement therapy, we gave 11 patients a total of 15 infusions of DDAVP (0.3 micrograms/kg IV) and measured the Simplate bleeding time pre- and postin-fusion. All patients were thrombocytopenic (one patient 100-150,000; seven 66-99,000; and three 50-65,000/microliters). Nine of 15 infusions resulted in at least a 2-min shortening of the bleeding time; in 6/11 infusions in which the platelet count was greater than 65,000, the bleeding time shortened to less than or equal to 10 min, in 1/11 it shortened greater than or equal to 2 min but not to less than or equal to 10 min. In only 2/4 infusions given to patients with platelet counts less than 65,000 did the bleeding time demonstrate any significant shortening. None of the six liver biopsies performed in the patients whose bleeding time shortened to less than or equal to 10 min resulted in any significant bleeding; blood products were not transfused either pre- or postprocedure. These limited data demonstrate that a high percentage of patients with platelet counts of 65-100,000/microliters can manifest a significant shortening of bleeding time following a standard infusion of DDAVP, and that in approximately half of these patients the bleeding time will shorten enough to safely allow the performance of a liver biopsy without the need for prophylactic transfusion of blood products.

Two methods of assessment of methotrexate hepatotoxicity in patients with rheumatoid arthritis.

Serial liver biopsy specimens from 18 patients with rheumatoid arthritis receiving a weekly dose of methotrexate 7.5-20 mg for a minimum of 12 months were assessed semiquantitatively and by a microcomputer image analysis system. The semiquantitative histological method showed a significant increase in pericellular collagen and in overall disease while morphometry showed a significant increase in pericellular, perivenular, and portal tract collagen. There was a significant correlation between the two methods, but morphometry had the advantage of objectivity and efficiency. There was no correlation between the increase in collagen and the accumulated dose of methotrexate, which suggests that other factors in addition to methotrexate may contribute to liver injury.

Serial liver biopsies are indicated with methotrexate therapy in juvenile RA

Serial liver biopsies are indicated with methotrexate therapy in juvenile RA

Morphometric assessment of hepatic fibrosis

A microcomputer image analysis system was used to measure collagen in sinus red stained liver biopsies:- normal liver n = 34, mild pericellular fibrosis n = 6, developing cirrhosis n = 8, micronodular cirrhosis n = 4. The amount of collagen in each biopsy was measured in optical density units. Compared with normal liver, optical density measurements were statistically significantly increased in the livers showing mild pericellular fibrosis (p 3 g; alcohol consumption up to 100 g/wk. Computerized measurement of these biopsies confirmed the progression of hepatic fibrosis as seen by routine light microscopy. Conclusion Computerised morphometry offers a rapid and sensitive method for assessment of hepatic fibrosis; this methodology is currently being applied to the evaluation of serial liver biopsies from patients on long-term methotrexate.

PROTECTIVE EFFECTS OF TRIFLUOPERAZINE ON THE MICROCIRCULATION OF COLD-STORED LIVERS

Previous studies have shown a protective effect of trifluoperazine (TFP), a calmodulin inhibitor, upon the microcirculation of cold-stored kidneys. The present study points to similar beneficial effects of TFP on the microcirculation of cold-stored livers; 25 canine livers were preserved for 24 hr with Euro-Collins' solution (EC) (n = 8), University of Wisconsin solution (UW) (n = 7), or UW + TFP (n = 10). The stored livers underwent heterotopic transplantation (HLTX); hepatic-artery and portal-vein pressure and flow were monitored; oxygen consumption and extraction were measured before HLTX and at 15-min intervals after reperfusion, for 1 hr. Mean hepatic-artery and portal-vein flow (HAF & PVF) prior to donor hepatectomy were 172 and 530 cc/min, respectively. Poor HAF and PVF occurred in EC-HLTX (mean 35, 175 cc/min, respectively). The damaged EC-flushed livers could not compensate to the decreased hepatic blood flow by increased oxygen extraction (oxygen consumption and extraction, 8.7 vol.% and 48%, respectively). Light and electron microscopy showed severe liver necrosis and periportal hemorrhages. Improved hepatic-artery and portal-vein flows were seen in UW HLTX (105 and 254 cc/min), and oxygen consumption and extraction were 16.4 vol.% and 66%, respectively. Liver biopsy taken just before reperfusion revealed well-preserved liver architecture. Liver biopsy obtained 1 hr after reperfusion revealed marked edema of the portal triad, sinusoid congestion, and hemorrhage. Electron-microscopy biopsies obtained during reperfusion at 15-min intervals revealed severe vasospasm of the terminal hepatic arterioles and progressive damage to the liver microcirculation. The addition of TFP to the UW-flush solution resulted in excellent protection of the liver microcirculation. Marked increase in hepatic-artery and portal-vein blood flow was noted after reperfusion (mean 167 and 421 cc/min, respectively (P 0.02 vs. UW: P 0.001 vs. EC). The recovery of metabolic activity was evident by the high oxygen consumption and extraction (25.8 vol.% and 80%, respectively). And serial liver biopsies obtained after reperfusion have shown excellent protection of liver architecture and the absence of hepatic arteriolar vasospasm. Taken together, these data suggest that the addition of TFP to the UW solution protects the liver microcirculation by rendering the hepatic microcirculation insensitive to vasospastic stimuli during reperfusion, thus permitting better metabolic recovery after transplantation.

Liver histology in patients receiving low dose pulse methotrexate for the treatment of rheumatoid arthritis.

The liver histology of 52 patients treated with intermittent low dose pulse methotrexate for rheumatoid arthritis was evaluated using a modification of the Roenigk grading system. Patients studied had had an average of 3.2 years of treatment or had received 1.7 g methotrexate. No patient had cirrhosis; 15 (29%) patients had evidence of mild fibrosis. Histological abnormalities were not predicted by liver function test changes, with the exception that hypoalbuminaemia occurred in 60% of those with grade IV (modified criteria) findings. The need for liver biopsy in patients with rheumatoid arthritis treated with methotrexate before two years or 1500 mg of treatment has not been established. Whether serial liver biopsies will be needed beyond this time has yet to be determined.

Characterization and Biological Properties of A Hepatitis B Virus Isolated From A Patient Without Hepatitis B Virus Serologic Markers

We have developed a rapid method to characterize genomic diversity of low-level hepatitis B and related viral agents after their identification in serum by high-affinity HBsAg-antibody monoclonal antibody capture and subsequent polymerase chain reaction amplification. Serum from an individual with chronic liver disease and without hepatitis B virus serological markers but reactive by monoclonal antibody capture/polymerase chain reaction amplification was inoculated into a chimpanzee. After inoculation, an acute hepatitis B virus-like hepatitis developed in the chimpanzee. Analysis of serial liver biopsy samples showed the persistence of hepatitis B virus DNA for more than 17 mo after resolution of acute hepatitis and seroconversion. Applying the technique of restriction enzyme fragment analysis to serial chimpanzee liver biopsy samples and acute-phase sera, along with the serum inoculum, we established that all hepatitis B virus DNA sequences are derived from the same viral agent. We present evidence that the viral DNA persisted as a nonreplicating episomal form in the nucleus of hepatocytes. This study demonstrates that after clinical and serological recovery from an acute hepatitis, there may be persistence of low-level hepatitis B virus-related genome in the liver despite the presence of antibodies to HBsAg. Such persistence of viral genome may be a natural sequela of infection and may serve as a source of viral latency and possible reactivation. Finally, cloning and complete nucleic-acid sequencing of this virus have demonstrated multiple nucleotide and amino acid changes compared with all known hepatitis B virus subtypes. These changes may have contributed in part to a different antigenic composition or immunological reactivity of the host to this hepatitis B virus isolate.

Intrahepatic Cholestasis and Phospholipidosis Associated With the Use of Trimethoprim–Sulfamethoxazole

Although liver injury after administration of the trimethoprim-sulfamethoxazole combination is rare, hepatocellular necrosis and cholestasis have developed in a few cases. We describe a patient who developed a severe, prolonged cholestatic reaction after trimethoprim-sulfamethoxazole administration. The findings from serial liver biopsy samples showed characteristic abnormalities of phospholipidosis that have not been previously described for trimethoprim-sulfamethoxazole-related hepatic injury. The most prominent finding on electron microscopic evaluation of the liver was the presence of prominent hepatocyte lysosomal inclusions characterized by concentric arrangements of membranous and lamellated structures. The patient improved after several courses of exchange plasmapheresis, which may have assisted in the removal of toxic drug-lipid complexes. The pathogenesis of this acquired secondary phospholipidosis is unknown. Possible mechanisms include generation of highly lipid-soluble metabolites and inhibition of the lysosomal enzyme phospholipase A1.

Chronic persistent hepatitis type B can be a progressive disease when associated with sustained virus replication

In 44 hepatitis B virus (HBV) carriers with chronic persistent hepatitis (CPH), serial liver biopsies were available. At presentation 38 patients had HBV-DNA in their serum including 31 HBeAg positive and seven anti-HBe positive cases. The remaining six patients were anti-HBe positive and HBV-DNA negative. During a mean histologic follow-up of 4.2 years, 12 (32%) of the 38 HBV-DNA positive patients progressed to chronic active hepatitis (six cases) or to active cirrhosis (six cases), while 26 patients showed either unchanged features of CPH (21 cases), or histologic improvement to normal liver (five cases). Persistence of HBV-DNA in serum, independently of HBeAg/anti-HBe events, was significantly ( p < 0.01) associated with deterioration of liver disease, while termination of HBV replication correlated significantly ( p < 0.05) with spontaneous biochemical remission and with unchanged or improved histology. None of the six anti-HBe positive patients without serologic markers of hepatitis B virus replication showed histologic deterioration. These findings indicate that continuing HBV replication is a marker which predicts unfavourable evolution of chronic persistent hepatitis and frequent transition to chronic active hepatitis or cirrhosis.

Further studies of 48‐1 antigen in serial liver biopsies from chimpanzees infected with hepatitis delta virus

The 48-1 antibody, initially reported to react specifically with non-A, non-B infected liver tissue, has been found to react also with liver specimens from chimpanzees infected with hepatitis delta virus (HDV). To clarify further the relation between HDV and appearance of the antigen reacting with the 48-1 antibody (48-1 Ag), immunoperoxidase studies were carried out on serial liver specimens from chimpanzees infected with HDV. Immunohistochemical and serological findings suggested that the appearance of 48-1 Ag paralleled that of HDV. Double immunoperoxidase staining revealed HDAg in the nucleus and 48-1 Ag in the cytoplasm of the same hepatocytes as well as in different hepatocytes separately. The course of appearance of microtubular aggregates paralleled that of 48-1 Ag. The present results suggested that expression of 48-1 Ag was related to infection with HDV, probably because expression of this antigen is induced from the host genome.

Semiquantitative Assessment of Cholestasis and Lymphocytic Piecemeal Necrosis in Primary Biliary Cirrhosis

Histologic activity of chronic cholestasis and lymphocytic piecemeal necrosis, a characteristic finding of chronic active hepatitis, was examined semiquantitatively in 157 liver biopsy specimens from 122 patients with primary biliary cirrhosis (PBC). Although both of these lesions were usually admixed variably in a single liver specimen, semiquantitative assessment made it possible to classify liver biopsy specimens into four groups: group A, no or minimum cholestatic or hepatitic changes (58 specimens); group B, predominantly cholestatic changes (37 specimens); and group C, predominantly hepatitic changes (54 specimens). Only eight specimens fell into group D, prominent cholestatic as well as hepatitic changes. Serial liver biopsies of specimens within groups B and C showed a persistence of group B- and C-type pathologies, while liver biopsies of group A specimens frequently changed to group B or C. Immunohistochemical studies illustrated that lymphocytic piecemeal necrosis mainly consisted of activated T lymphocytes as seen in chronic active hepatitis. Our data suggest that either of two hepatic parenchymal lesions predominates and persists in each liver biopsy specimen. A high cholestatic score appeared to relate to poor prognoses of the patients and also to the degree of cirrhotic transformation. This grouping system may be valuable in the clinicopathologic assessment of PBC, when it is combined with ordinary staging.

Methotrexate-Induced Cirrhosis Requiring Liver Transplantation in Three Patients With Psoriasis

Methotrexate has been used for many years to treat refractory psoriasis. Three cases of methotrexate-induced cirrhosis requiring orthotopic liver transplantation are presented to emphasize the importance of strict adherence to published criteria for patient selection, monitoring of cumulative drug dosages, and the performance of serial liver biopsies. Each patient had been treated with long-term methotrexate therapy (cumulative doses far in excess of 1.5 g) without undergoing serial liver biopsies, contrary to well-established treatment guidelines. Caution must be exercised in using methotrexate as a steroid-sparing agent in the treatment of inflammatory diseases because of its potential to cause severe hepatotoxic effects with long-term usage and cumulative doses above 1.5 g. Patients easily become psychologically dependent on the drug, and physicians need to guard against the false sense of security engendered by normal results on liver function studies.

Two methods of assessment of methotrexate hepatotoxicity

Methotrexate (MTX) hepatoxicity was evaluated in serial liver biopsies from 18 patients with rheumatoid arthritis receiving weekly MTX 7.5-20mg for a minimum of 12 months. Fatty change, focal necrosis, nuclear pleomorphism, Kupffer cell enlargement and hepatic fibrosis were assessed using a semiquantitative grading system; the results were compared to those obtained using a microcomputer analysis system to measure changes in hepatic collagen. Subjective assessment showed a small but significant increase in overall pathology (p<0.02) and pericellular collagen (p<0.01), while morphometry showed a significant increase in pericellular (p<0.02) perivenular (p<0.05) and portal tract (p<0.05> collagen. <h3>Conclusion</h3> Computerised morphometry offers a rapid, reliable and sensitive method for the assessment of progressive hepatic fibrosis occurring in association with MTX.