Iron overload in patients with chronic hepatitis C: A clinicopathologic study

Iron Overload in Patients With Chronic Hepatitis C: A Clinicopathologic Study S, HAQUE, MD, B. CHANDRA, MD, M, A, GERBER, MD, AND A. S. F. LOK, MD Recent studies suggest that increased hepatic iron may impair the response to interferon therapy in patients with chronic hepatitis C. We reviewed the records and liver biopsies of 72 patients with chronic hepatitis C to determine the prevalence of iron overload and to evaluate whether there is a correlation between serum and hepatic iron concentrations and activity of liver disease. Patients with other causes of liver disease or iron overload were excluded. Necroinflammatory activity and fibrosis were evaluated using modified Knodell score. Hepatic iron was assessed using Brissot's grading system. Increased serum iron and ferritin levels were found in 29% and 43% patients, respectively. Hepatic iron grades 0, I, II, III, and IV were present in 37%, 35%, 25%, 3%, and 0% of patients, respectively. A significant correlation was found between hepatic iron grade and

serum ferritin (P = .0001). There was no correlation between hepatic iron grade and histological activity index or fibrosis score. In stunmary, we found a high proportion of patients with chronic hepatitis C had mild to moderate increase in hepatic iron content even when patients with alcoholism and recurrent transfusions were excluded. However, very few patients had severely increased iron load. HUM PATrlOL 27:1277-1281. Copyright © 1996 by W.B. Samlders Company Key words: chronic hepatitis C, hepatic iron, ferritin, alpha interferon, histological activity index. Abbreviations: ALT, alanine anlinotransferase; HCV, hepatitis C virus; TIBC, total iron-binding capacity; TF sat, transferrin saturation; HAI, histological activity index.

A l p h a i n t e r f e r o n is t h e o n l y a p p r o v e d t r e a t m e n t for c h r o n i c h e p a t i t i s C. H o w e v e r , o n l y a p p r o x i m a t e l y 40% o f t r e a t e d p a t i e n t s r e s p o n d with n o r m a l i z a t i o n in s e r u m a l a n i n e a m i n o t r a n s f e r a s e (ALT) levels at t h e e n d o f a 6 - m o n t h c o u r s e o f t r e a t m e n t . 1 Several factors have b e e n r e p o r t e d to b e a s s o c i a t e d with n o n r e s p o n s e to i n t e r f e r o n t h e r a p y , 2'3 o n e o f t h e s e factors is h i g h hepatic i r o n c o n c e n t r a t i o n . 4'5 I r o n is a n i m p o r t a n t n u t r i e n t for m i c r o b i a l growth. I r o n o v e r l o a d has b e e n s h o w n to i m p a i r t h e i m m u n e r e s p o n s e . 6-9 Several investigators also r e p o r t e d t h a t thalassemics with h i g h e r h e p a t i c i r o n c o n c e n t r a t i o n have m o r e active liver disease ~°'~t a n d lower r e s p o n s e rates to i n t e r f e r o n t h e r a p y . 12 Thus, h i g h h e p a t i c i r o n c o n c e n t r a t i o n m a y d e c r e a s e r e s p o n s e to i n t e r f e r o n t h e r a p y by e n h a n c i n g h e p a t i t i s C virus (HCV) r e p l i c a t i o n o r by a g g r a v a t i n g H C V - i n d n c e d liver d a m a g e . This has l e d to clinical studies e v a l u a t i n g t h e r o l e o f p h l e b o t o m y as a t r e a t m e n t f o r c h r o n i c h e p a t i t i s C. x3 H o w e v e r , t h e r e is as y e t very little d a t a o n t h e prevalence, d e g r e e , a n d m e c h a n i s m s o f i r o n o v e r l o a d in patients with c h r o n i c h e p a t i t i s C, p a r t i c u l a r l y t h o s e w h o d o n o t have c o n c o m i t a n t factors such as excessive alcoh o l c o n s u m p t i o n a n d r e c u r r e n t transfusions t h a t cont r i b u t e to i n c r e a s e d h e p a t i c i r o n c o n t e n t . T h e aims o f this study were to d e t e r m i n e t h e preval e n c e o f i r o n o v e r l o a d in c h r o n i c h e p a t i t i s C p a t i e n t s w h o d o n o t have a n y o t h e r k n o w n factors t h a t m a y c o n t r i b u t e to i r o n o v e r l o a d , a n d to d e t e r m i n e if t h e r e is a c o r r e l a t i o n b e t w e e n s e r u m / h e p a t i c i r o n c o n c e n t r a tion a n d b i o c h e m i c a l a n d h i s t o l o g i c a l a s s e s s m e n t o f t h e activity a n d stage o f H C V - i n d u c e d liver disease.

MATERIALS AND METHODS

From the Department of Pathology and Laboratory Medicine and the Department of Medicine, Section of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, LA; and the Division of Gasn-oenterology, University of Michigan Medical Center, Ann Arbor, MI. Accepted for publication May 3, 1996. Address correspondence and reprint requests to Salima Haque, MD, Department of Pathology and Laboratory Medicine, Tulane University Medical Center, 1430 Tulane Ave, New Orleans, LA 70112. Copyright © 1996 by W.B. Saunders Company 0046-8177/96/2712-002655.00/0

Consecutive liver biopsies performed on hepatitis C antibody (anti-HCV)-positive patients at the Tulane University Medical Center between 1990 and 1995 were reviewed. Liver biopsy specimens from patients who had concomitant causes of liver disease or iron overload, including hepatitis B surface antigen positivity, alcoholism, and requirement for recurrent transfusions; patients who had received interferon therapy before the biopsies; and those who had inadequate-size biopsy specimens, were excluded from the analysis. All of the biopsy specimens were formalin fixed and paraffin embedded. Five were wedge biopsies, and the rest were needle biopsies. The patient records were reviewed to confirm the cause of liver disease, to rule out other causes of iron overload, and to verify current medications and previous interferon therapy. Serum ALT, iron, total iron-binding capacity (TIBC), percentage transferrin saturation (TF sat), and ferritin values within 1 month of the biopsies were recorded. Anti-HCV was tested by" second-generation enzyme immunoassay (Abbott Laboratories, North Chicago, IL). Serum iron, TIBC, TF sat, and ferritin were tested by colorimetric method (BoehringerMannheim, Indianapolis, IN). Slides stained with hematoxylin-eosin, trichrome, reticulin, and Perl's Prussian blue from each patient were reviewed. The activity of the necroinflammatory process was graded from 0 to 18 using the first three components of the Knodell histological activity index (HAl). TM Fibrosis or stage of the liver disease was evaluated separately and scored from 0 to 4 (Table 1). Hepatic iron was assessed using Brissot's l~ semiquantitatire histological grading system. This system takes into account not only iron in hepatocytes but also iron in Kupffer cells and fibrous tracts. Heavier weight is given to iron in hepatocytes. Iron in hepatocytes, Kupffer cells, and fibrous tracts was separately scored from 0 to 4 based on the percentage of cells with stainable iron as well as the quality of the iron deposit. These scores were then multiplied by a coefficient of 3 for iron in hepatocytes, and a coefficient of 1 for iron in Kupffer cells and fibrous tracts, giving rise to an iron score of 0 to 12 for hepatocytes, and 0 to 4 for Kupffer cells and fibrous tracts, and a total hepatic iron score of 0 to 20. Hepatic iron grade was derived from the total hepatic iron score: with scores of 0 being grade 0, 1 to 5 being grade I, 6 to 10 being

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HUMAN PATHOLOGY Volume27, No, 12 (December 1996) TABLE 1.

Modified Knodell Score

Clinical Features of 72 Chronic

TABLE 2.

Hepatitis C Patients Histological Activity I n d e x (HA/)

Fibrosis Score (Staging)

Range of Score

Component

Score

Description

0-10 0-4 0-4

Periportal necrosis or bridging necrosis Lobular activity Portal inflammation

0 1 2 3 4

No fibrosis Mild fibrosis Moderate fibrosis Severe fibrosis Cirrhosis

Number Age in years ALT U / L Normal value M < 41; F < 35 Ferritin ( n g / m L ) Normal value M 14-179 F 5-146 S e r u m Iron ( # g / d L ) Normal value 48-160 % Saturation Normal value

grade II, 11 to 15 being grade III, and 16 to 20 being grade IV. Spearman's nonparametric test was used to calculate the linear correlation coefficients.

Male

Female

36 44.42 _+ 1.75" 18-70 t 144.39 _+ 22.28* 16-682t

36 52.22 -+ 1.90" 28-77 t 123.53 + 14.79" 12-411 t

552.18 _+ 132.05"

261.06 + 49.38*

13-2775 t

23-791t

110.47 + 14.25' 19-253 t

126.12 + 13.47" 42-207 t

40.26 -+ 5.8* 8-101 t

42.67 ± 6.25* 11-98 t

20-50 Hepatic iron score Normal value 0-Trace

RESULTS Liver biopsy specimens f r o m 72 patients were included in this study. T h e r e were 36 m e n and 36 women, with a m e a n age of 48 + 1 years (range, 18 to 77). T h e m e a n serum ALT level at the time of biopsy was 134 _+ 13 U / L (range, 12 to 682 U / L ) .

3.33 +-_ * 0-12t

* Mean _+ SEM. t Range.

Histological Activity a n d Stage

Serum Iron Indices Forty (56%) patients had serum ferritin levels within 1 m o n t h of the liver biopsies. A m o n g these, 34 also had simultaneous serum iron and TF sat values, all within 1 m o n t h of the liver biopsies. T h e r e was no difference in sex, age, activity and stage of liver disease, and hepatic iron score between this subset of patients and those in w h o m serum iron indices were not available. A m o n g the 40 patients who had serum ferritin values within 1 m o n t h of the liver biopsies, 17 (43%) had ferritin values above the u p p e r limit of normal, and eight (20%) had ferritin values in the h e m o c h r o m a t o t i c range ( > 6 5 0 n g / m L ) . T h e m e a n serum ferritin value was 421 + 79 n g / m L (range, 13 to 2,775 n g / m L ; normal for male patients: 14 to 179 n g / m L and female patients: 5 to 146 n g / m L ) (Table 2). O f the 34 patients who h a d serum iron and TF sat values within 1 m o n t h of the liver biopsies, 10 (29%) had elevated serum iron levels, and six (18%) had TF sat in the h e m o c h r o m a t o t i c range ( > 6 2 % ) . T h e m e a n serum iron level was 120 + 10 ]zg/dL (range, 19 to 253 # g / d L ; normal, 48 to 160 # g / d L ) , and the m e a n TF sat was 41% _+ 4% (range, 8% to 100%; normal, 20% to 50%) (Table 2). A m o n g the 34 patients who h a d all the serum iron indices within 1 m o n t h of the liver biopsies, eight (24%) had TF sat and serum ferritin values that exceeded the u p p e r limit of normal, and two (6%) had TF sat and ferritin values that were b o t h within the h e m o c h r o m a totic range. T h e m e a n serum ferritin levels were higher in m e n than in w o m e n (552 n g / m L v 261 n g / m L , P = .05), but there was no difference in serum iron levels or TF sat between the two sexes.

3.33 + 0.59* 0-15t

T h e m e a n HA1 was 4.7 _+ 1.6 (range, l to 8; minimal to mild histological activity), whereas the m e a n fibrosis score was 3.1 +_ 0.1 (range, 1 to 4; mild fibrosis to cirrhosis). Thirty-five (49%) patients had established cirrhosis. Histological features that are characteristic of hepatitis C were f o u n d in most of the patients: lymphoid aggregates (81%), bile duct damage (97%), bile ductular proliferation (69%), and steatosis (43%).

Hepatic Iron Content a n d Distribution of Hepatic Iron Twenty-seven (37%) patients had no stainable iron in their liver biopsy specimens; of these, 13 (48%) h a d stage 4 fibrosis or cirrhosis (Fig 1). Twenty-five (35%)

40 35, 30' 25, 201

"6 15, .O

E

10' 5, O" 0

1

II

III

Hepatic Iron Grade

l l~l Total cases FIGURE I .

[] Kupffer cells [] Hepatocytes [] Mixed

Hepatic iron

with chronic hepatitis C.

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grade and distribution in 72 patients

I

CHRONIC HEPATIC C AND HEPATIC IRON (Haque et al)

and ALT levels (P = .0014), but there was no correlation between hepatic iron grade and sex, age, serum iron, TF sat, HA[, or fibrosis score. Significant correlations were found between hepatic fibrosis score and age (P < .0001) and HA1 (P = .001), but there was no correlation between hepatic fibrosis score and serum ALT level or s e r u m / h e p a t i c iron content. T h e r e was no correlation between HA1 and serum ALT level, serum iron indices, or hepatic iron content. T h e r e was no correlation between serum ferritin levels and biochemical (ALT level) or histological (inflammatory) activity index (HA/).

DISCUSSION In this study, we f o u n d that a high proportion (30% to 40%) of patients with chronic hepatitis C had increased serum iron and ferritin levels even after exclusion of patients who required recurrent transfusions. Increased serum ferritin levels may be observed in patients with chronic inflammation. However, there was no correlation between serum ferritin level and in-

FIGURE 2. Grade I stainable iron in the liver of a patient with chronic hepatitis C, Note that the iron is confined to Kupffer cells only (arrows). This staining paffern was present in 68% of our patients with grade I hepatic iron. (Perl's Prussian blue stain; original magnification x570.)

patients had grade 1 stainable iron in their liver biopsy specimens. Of these, 17 (68%) had stainable iron in the Kupffer cells and fibrous tracts only (Fig 2), two (8%) had iron in the hepatocytes only (Fig 3), and the remaining six (24%) had iron in the Kupffer cells, fibrous tracts as well as hepatocytes. Twelve (48%) of these patients had cirrhosis. Eighteen (25%) patients had grade II stainable iron in their liver biopsy specimens. Iron was uniformly distributed in the hepatocytes, Kupffer cells, and portal tracts, except for one patient whose hepatic iron was confined to the hepatocytes only. Nine (50%) of these patients had cirrhosis. Two (3%) patients had grade III stainable iron in their liver biopsy specimens. Both patients had cirrhosis. Iron was present in the hepatocytes, Kupffer cells, and portal tracts. There was a p r e p o n d e r a n c e of iron within the periportal hepatocytes (Fig 4). None of the patients had grade IV stainable iron in their liver biopsy specimens. There was no difference in the hepatic iron content for the two sexes, mean hepatic iron score being 3.3 for both male and female patients. Correlation Between Serum/Hepatic Iron Content and Histological Activity/State of Liver D i s e a s e

Significant correlations (Table 3) were found between hepatic iron grade and serum ferritin (P = .0001)

FIGURE 3. Grade I stainable iron in the liver of a patient with chronic hepatitis C. The iron is present only within the hepatocytes (arrows), This pattern was seen in 8% of patients with grade I hepatic iron. (PeWs Prussian blue stain; original magnification x570.)

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Volume 27, No. 12 (December 1996)

FIGURE 4. Grade III stainable iron in the liver of a patient with chronic hepatitis C. The iron is present within hepatocytes, Kupffer ceils, and fibrous tract. Note that the iron in hepatocytes is predominantly in a periportal distribution. (Perl's Prussian blue stain; original magnification x190,)

flammatory activity of liver disease as assessed biochemically (ALT level) and histologically (HAI). Iron is normally stored in the liver in the form of ferritin and hemosiderin, predominantly in the hepatocytes. ~6 Ferritin is water soluble and histochemically not stainable. Hemosiderin is insoluble and histochemically stainable. Hemosiderin is normally a minor c o m p o n e n t of hepatic iron, but becomes the major form of storage iron with increasing iron overload. Iron in Kupffer cells in usually caused by accumulation of ferritin and hemosiderin released from damaged tissues or during cell turnover. Normal liver has no or only trace a m o u n t of stainable iron in hepatocytes. 17 We found that 63% of our chronic hepatitis C patients had grade I or more hepatic iron even through we excluded patients who may have other causes of increased hepatic iron: alcoholics and those who required recurrent transfusions. Hepatic iron is thought to increase with age and in patients with cirrhosis. However, we f o u n d no correlation between hepatic iron grade and age or fibrosis score.

There was a significant correlation between hepatic iron grade and serum ferritin level but not serum iron 1280

level or TF sat. Similar results have been found by other investigators. 15'ts Our study suggests that serum ferritin level may be an indirect parameter for assessing hepatic iron load. However, other investigators found that response to interferon therapy correlated with hepatic iron concentration but not with serum ferritin level. 4 Hepatic iron appeared to preferentially accumulate in Kupffer cells, at least initially. O f the 25 patients who had grade I stainable iron in their liver biopsy specimens, 68% had iron in the Kupffer cells and portal tracts only, whereas iron was uniformly distributed in the hepatocytes, Kupffer cells, and portal tracts in patients who had grade II or III stainable iron. The mechanism for the accumulation of iron in patients with chronic hepatitis C is not known. It is possible that chronic hepatitis causes increased turnover of hepatocytes resulting in increased release of iron, which is taken up by Kupffer cells. As the hepatic iron content increases, some of it also accumulates in hepatocytes. We f o u n d a significant correlation between hepatic iron grade and ALT levels but not HAl. Because HAI is a better indicator of hepatic inflammation over time, other mechanisms may be involved in the iron accumulation. Hepatitis C virus infection may affect iron metabolism in the reticuloendothelial system, resulting in iron accumulation, as has been suggested in patients with chronic hepatitis B virus infections. ~9'2° Furthermore, the lack of any correlation between HA[ and liver iron content suggests that hepatic iron concentration may not have any direct influence on the severity of the necroinflammation. Significant association was f o u n d between HAI and hepatic fibrosis, probably reflecting the persistence of necroinflammatory activity into the late stages of the disease. Although a high proportion of our patients had mild to moderately increased hepatic iron content, very few had severe iron overload. Only 6% of the patients studied had TF sat and ferritin values that were both within the hemochromatotic range. N o n e of the patients had grade IV and only 3% had grade III hepatic iron. Increased serum iron and ferritin levels in the absence of increased hepatic iron content has been observed by other investigators. 21 In addition, studies that reported increased hepatic iron concentration in TABLE 3. C l i n i c o p a t h o l o g i c a l Correlations Between Serum/Hepatic Iron C o n t e n t a n d Hepatic Inflammation/Fibrosis Correlations

rho

P

Hepatic iron v serum ferritin Hepatic iron v ALT Hepatic iron v HAI Hepatic iron v fibrosis Hepatic iron v age Hepatic fibrosis v age Hepatic fibrosis v HAI Hepatic fibrosis v ALT Hepatic fibrosis v serum ferritin HA1 v ALT HAI v serum ferritin Serum ferritin v ALT ALT v serum iron

0.56 0.36 0.01 0.07 0.02 0.44 0.37 0.20 0.23 0.19 0.03 0.03 0.45

.000I .0014 .8803 .5394 .8674 <.000i .0011 .0883 .1424 ,1070 ,8379 ,8527 .0070

CHRONIC HEPATIC C AND HEPATIC IRON (Haque et al)

nonresponders to interferon therapy found that only 10% of chronic hepatitis C patients had hepatic iron concentration above the u p p e r limit of normal. 4'5 Thus, severe increase in hepatic iron content is rare in patients with chronic hepatitis C. The finding of excessive hepatic iron ( > g r a d e II1) in patients with chronic hepatitis C should raise suspicion of coexistent genetic hemochromatosis. Hemochromatosis is one of the commonest inherited metabolic disorder, affecting approximately 1 in 250 Americans of North European extraction. 22 The close correlation between chemical and histochemical liver iron content has been well established by previous s t u d i e s . 15'17'2~-25Liver biopsy in chronic hepatitis C may play an important role not only in evaluating the inflammatory activity and extent of fibrosis, but also in assessing hepatic iron store and distribution. It could be speculated that the distribution of hepatic iron, especially Kupffer cell iron deposition, may eventually turn out to be one of the indicators of therapeutic response to interferon alpha. In summary, we found that a high proportion of patients with chronic hepatitis C had increased serum and hepatic iron content even when patients with alcoholism and recurrent transfusions were excluded. There was a good correlation between serum ferritin level and hepatic iron content. The mechanisms of iron accumulation in patients with chronic hepatitis C is not clear but appears to occur in the Kupffer cells initially. In accordance with other investigations, we f o u n d that very few patients had severely increased hepatic iron. Thus, the role of phlebotomy in the treatment of chronic hepatitis C remains to be determined.

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