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Journal of Pediatric Gastroenterology & Nutrition:
doi: 10.1097/MPG.0000000000000235
Original Articles: Hepatology and Nutrition

Ferritin and LIC: Predicting Liver Injury in Children With Sickle Cell

Smith, Erika*; Lebensburger, Jeffrey*; Hilliard, Lee*; Kelly, David*; Fineberg, Naomi*; Bai, Shuting; Howard, Tom*

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Author Information

*University of Alabama at Birmingham

NYU Langone Medical Center, New York, NY.

Address correspondence and reprint requests to Erika Smith, MD, Pediatric Gastroenterology Fellow, Pediatric Gastroenterology and Nutrition, Children's of Alabama, 1600 7th Avenue South, Lowder 618, Birmingham, AL 35233 (e-mail: esmith@peds.uab.edu).

Received 1 July, 2013

Accepted 29 October, 2013

The authors report no conflicts of interest.

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Abstract

Objective: Chronic blood transfusion therapy reduces clinical events in children with sickle cell anemia but increases risk for an iron-related liver injury. Liver biopsy is the gold standard technique for quantifying liver iron content (LIC) and evaluating liver pathology. Ferritin, liver enzymes, and R2* magnetic resonance imaging of the liver are obtained as surrogate markers. In this study we compared surrogate markers with the gold standard, liver biopsy, in assessing liver histology.

Methods: We conducted a retrospective review of 259 liver biopsies in 109 children with sickle cell anemia on chronic transfusion therapy and chelation therapy during a 9-year period at a single center. Liver pathology was compared with LIC, ferritin, and alanine aminotransferase.

Results: Ferritin correlates with LIC (r = 0.74, P < 0.001), although there is a broad range of ferritin values for a given LIC. Furthermore, patients with a high LIC (≥7 mg Fe/g dry weight) demonstrated significantly higher ferritin as compared to the patients with lower LIC <7 (P < 0.001). Periportal/portal inflammation also showed a significant relation. There was no significance when comparing ferritin and lobular inflammation or ferritin and alanine aminotransferase. When evaluating LIC in relation to fibrosis, the present study revealed that there was only a significant correlation with severe fibrosis (F = 36, P < 0.001).

Conclusions: The results suggest that although correlations exist among ferritin and LIC and severe fibrosis and LIC, caution should be taken when they are used in isolation. Liver biopsy provides important pathologic information that cannot be obtained through surrogate markers.

Patients with sickle cell anemia can experience frequent clinical complications, including vaso-occlusive disease, dactylitis, acute chest syndrome, and stroke. Extensive research in primary and secondary prophylaxis for stroke victims has shown that chronic transfusions are beneficial for these “at-risk” patients with sickle cell. Patients deemed at risk for stroke include those with an abnormal transcranial Doppler ultrasound, as well as those who have already experienced a stroke (1,2). Although chronic transfusion decreases stroke and other clinical events associated with sickle cell hemoglobinopathy, this therapy is also associated with its own risks, including red cell alloimmunization, infection, transfusion reactions, and iron overload. Alloimmunization and bacterial or viral contamination have been reduced with improved blood banking techniques. Iron overload reduction can be accomplished through exchange transfusions or depletion strategies, including chelation therapies (3). The goal of chelation therapy is to bind nontransferrin bound iron (NTBI) and excess iron in cells, so that it can be excreted from the body. Unfortunately, poor adherence to chelation therapy or lack of access to red cell erythrocytapheresis perpetuates iron overload (4); this remains a relevant topic for discussion.

To better manage patients with iron overload, it is important to understand the physiology of iron uptake. The eventual destruction of aged erythrocytes (whether native or transfused) and hemoglobin degradation take place in the macrophages of the liver, bone marrow, and spleen. Hemoglobin is digested and iron is released bound to transferrin. The remaining iron enters the storage pool as ferritin or hemosiderin. As a patient receives successive blood transfusions, the storage capacity of the macrophages, however, increases releasing excess iron into circulation where it is taken up by transferrin. As transferrin's threshold is exceeded, excess iron may be stored in the hepatocytes. Over time, even the hepatocytes reach their maximum capacity for iron and release it back into plasma, where it becomes NTBI. NTBI is significant because this is a labile form of iron that is cytotoxic to the liver, heart, and pancreas (5). Over time, the NTBI produces inflammation and eventual fibrosis. Investigations have found that the patients with sickle cell disease tend to have less damage to the heart and other extrahepatic organs when compared with the patients with thalassemia. Patients with sickle cell tend to have an increased daily iron excretion, which may explain the decrease in cardiac damage. Inati et al (6) hypothesized that this is the result of increased intravascular hemolysis, hemefiltration, and increased renal absorption among this population.

For the children receiving recurrent transfusion therapy, clinicians must develop a plan to monitor the iron overload. The present surveillance methods for iron overload include measuring serum ferritin, alanine aminotransferase (ALT), NTBI, liver biopsy, and R2* magnetic resonance imaging (MRI). Ferritin is a serum marker of intracellular iron deposition. It is easily measured and physicians can trend it to evaluate the iron overload. Unfortunately, variation can exist in ferritin because it is also an acute-phase reactant leading to fluctuations in the level (7–11). Ho et al (12) recommended evaluating ferritin during a period of time rather than just an isolated level to allow for a trend with more significant meaning in interpretation. A study in adults by Inati et al compared the total number of transfusions to serum ferritin concentration and LIC, as well as the transfusion rate to ferritin and LIC. They noted a linear relation while comparing transfusion rate with ferritin and LIC. Other studies, however, have not been able to replicate this (6,13). Liver biopsy, the gold standard for iron overload, allows for a direct measure of hepatic iron content and assessment of histopathologic changes. This, however, is more invasive than venopuncture, with risks of bleeding, cost of extensive postoperative monitoring, and the potential for introducing infection. Another limitation is that liver iron deposition is not homogenous, and variation can exist among sample sites. Several studies have attempted to determine the most efficacious way to appropriately evaluate liver injury in patients on chronic transfusions with no definitive conclusions determined.

An emerging popular alternative is R2* MRI. The evidence suggests that MRI correlates well with liver iron content (LIC) obtained from a liver biopsy (14–16). Studies have shown that this may be equivalent to a liver biopsy in measuring LIC (17). MRI is noninvasive and allows for visualization of the entire liver as compared to a liver biopsy that only represents a sample of the liver. This is beneficial because iron deposition is heterogeneous within the liver. There are downsides to MRI as well, including sedation requirements for some patients so that they remain still for the duration of the study. To our knowledge, no study has compared MRI iron content with liver fibrosis. This concept is important because it emphasizes that LIC is an indirect surrogate for the measurement of histopathologic change.

The aim of our study was to identify the correlation of ferritin and LIC with liver histopathology in a large cohort to accurately assess the utility of serum markers as the indicators of hepatic injury in patients with serial transfusions.

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METHODS

We performed a retrospective review of 259 liver biopsies performed in 109 children on chronic transfusion therapy during a 9-year period. The study dates were September 2002 through December 2010. Institutional review board approval was obtained. The inclusion criteria consisted of patients between 0 and 30 years (of which only 4 patients were older than 21 years at first biopsy) with chronic transfusions for sickle cell disease.

Blood samples were required at the time of liver biopsy for laboratory tests and included ALT, aspartate aminotransferase, bilirubin, prothrombin time, partial thromboplastin time, and ferritin. Chelation with deferoxamine or deferasirox was determined by the physician with a practice standard of initiation of chelation once ferritin increased to >1000 ng/mL. All of the patients at the time of liver biopsy were treated with chelation therapy with either deferoxamine (30%) or deferasirox (70%). There was no incentive to be included in the study.

Liver histological sections were evaluated by 2 pathologists for the presence of portal/periportal inflammation, lobular inflammation, and fibrosis without knowledge of the patient's clinical course. Portal/periportal and lobular inflammation were scored 0 through 2, ranging from no inflammation to severe inflammation, respectively. Fibrosis was graded as negative (0), mild (1), moderate (2), and severe (3) (18).

The t tests were used to compare nominal data (ferritin and LIC and ferritin and ALT). The analysis of variance (ANOVA) was used for categorical data to evaluate and compare ferritin with the degrees of portal/periportal inflammation, lobular inflammation, and fibrosis. If the ANOVA was significant, the Tukey honestly significant difference test was performed to further evaluate the differences. Pearson correlation coefficients were used to determine the linear relations between the variables. Data were determined significant with a P value <0.05. All calculations were made using SPSS version 20 (SPSS Inc, Chicago, IL).

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RESULTS

Ninety-one patients had sickle cell hemoglobinopathy, 17 had sickle cell-β thalassemia, and 1 patient had sickle cell-D hemoglobinopathy. Chronic transfusion therapy was performed by either simple transfusion (65%) or erythrocytapheresis (35%) and was primarily for stroke prevention (n = 234). A few patients (n = 22) received transfusions for other indications, including a history of acute chest syndrome, restrictive pulmonary disease, and acute vascular necrosis. Three patients did not have information regarding indication for chronic transfusions. Patients were initiated on chronic transfusion at a mean age of 6.2 ± 3.6 years (0.75–17 years) with a liver biopsy obtained at a mean age of 14.6 ± 5.3 years (3–34 years).

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Ferritin and LIC

The mean serum ferritin level was 3509 ± 2617 ng/mL (range 64–13,576 ng/mL) and the mean LIC was 17.12 ± 12.98 mg Fe/g dry weight (range 0.14–53.21 mg Fe/g dry weight). We found a linear relation between ferritin concentration and LIC (r = 0.74, P < 0.001), but a spread was present (R2 = 55%) (Fig. 1). An estimate of LIC can be calculated by the equation LIC = 4.36 + 0.004 × ferritin. We did find 2 outliers that did not follow the predicted calculation. One patient with a ferritin of 3734 ng/mL had a predicted LIC of 17.92 mg Fe/g dry weight when the actual LIC was 53.21 mg Fe/g dry weight. This example shows the limitation in using this linear relation for an individual prediction. We also compared ferritin mean values for when LIC ≥ 7 or LIC < 7, a generally accepted value of concern for liver injury. The mean ferritin for LIC < 7 mg Fe/g dry weight was 1468 ± 1344 ng/mL and the mean for LIC ≥ 7 mg Fe/g dry weight was 3397 ± 2579 ng/mL (P < 0.001). Figure 2 presents a box plot comparison of the 2 groups. Receiver-operating characteristic curves (ROC) were created to evaluate the sensitivity and specificity of ferritin that would identify an LIC ≥ 7 mg Fe/g dry weight. (See Table 1 for sensitivity and specificity, and Fig. 3 for ROC.)

Figure 1
Figure 1
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Figure 2
Figure 2
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Table 1
Table 1
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Figure 3
Figure 3
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Ferritin and Liver Biopsy
Equation (Uncited)
Equation (Uncited)
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Periportal inflammation was graded as 0 in 129 biopsies, grade 1 in 88 liver biopsies, and grade 2 in 38 liver biopsies. Four biopsies did not have a ferritin level drawn at the time of the biopsy. The ferritin levels correlated with the grading of periportal inflammation (F = 21, P < 0.001 for 0 < 1 < 2); however, an overlap existed between the groups. The mean ferritin concentration for grade 0 periportal inflammation was 2700 ± 2016 ng/mL, grade 1 periportal inflammation was 3888 ± 2514 ng/mL, and grade 2 periportal inflammation was 5511 ± 3378 ng/mL. Lobular inflammation was graded as 0 in 28 liver biopsies, grade 1 in 223 liver biopsies, and grade 2 in 4 liver biopsies. No statistical differences were detected for lobular inflammation (F = 1.6, P = 0.198). Fibrosis was graded as 0 in 23 liver biopsies, grade 1 in 104 liver biopsies, grade 2 in 102 liver biopsies, and grade 3 in 26 liver biopsies. The mean serum ferritin concentration for grade 0 fibrosis was 2584 ± 1314 ng/mL, grade 1 fibrosis was 2626 ± 2071 ng/mL, grade 2 fibrosis was 3645 ± 2222 ng/mL, and grade 3 fibrosis was 7519 ± 3117 ng/mL. Individuals with grade 3 fibrosis had a significantly higher serum ferritin concentration, but no differences were detected with lower grades (Table 2).

Equation (Uncited)
Equation (Uncited)
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LIC and Liver Biopsy

Of interest, we looked at LIC and compared it with liver fibrosis to determine whether there was a direct correlation between the 2 variables. We found that a fibrosis score of 0 had a mean LIC of 13.3 ± 10.5 mg Fe/g dry weight (n = 22), a fibrosis score of 1 had a mean LIC of 12.6 ± 10.5 mg Fe/g dry weight (n = 102), a fibrosis score of 2 had a mean LIC of 18.8 ± 12 mg Fe/g dry weight (n = 101), and a fibrosis score of 3 had a mean LIC of 31.9 ± 11.6 mg Fe/g dry weight (n = 26). Eight liver biopsies did not have an associated LIC. The statistical analysis showed that there was no difference when comparing LIC for fibrosis score 0, 1, or 2. A fibrosis score of 3, however, was statistically different (Table 3).

Table 2
Table 2
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Ferritin and ALT

We also compared ferritin and ALT to evaluate the relation between the 2 serum markers. From the data, a regression equation was found: ALT = 25.17 + 0.004 × ferritin (P < 0.001). Unfortunately, the relation was weak, with a correlation coefficient of only 8%.

Table 3
Table 3
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DISCUSSION

Chronic blood transfusion therapy reduces clinical events and prevents recurrent brain ischemia in children with sickle cell anemia. The benefit of administering recurrent transfusions is weighed against the risk of an increased iron burden leading to chronic end-organ damage. To better assess the iron overload, we evaluated the surrogate markers for histopathologic change. The present study provides insight into the association between numerous factors used to evaluate iron overload in patients with sickle cell on chronic transfusion. Although there have been inconsistent data regarding the relation between ferritin and LIC, the present study shows a linear relation between the 2 factors that is statistically significant. Although variation does exist among individual data points, this linear relation can help clinicians determine when to start chelation therapy or when to adjust dosing. For example, in reviewing the data, to estimate an LIC ≥ 7 mg Fe/g dry weight by evaluating ferritin levels, a ferritin level would on average be 3397 ± 2579 ng/mL. Although this gives physicians guidance regarding the severity of liver disease, there is a large range and thus caution should be used. The ROC highlighted in the present study shows how to use various ferritin cutoff values to attempt to show the probability that LIC will be <7 or ≥7, which can be used clinically to assist physicians in the medical intervention of their patients.

Previous studies have evaluated the different factors in determining liver fibrosis. MRI has become a focal point because it can evaluate LIC without being invasive, unlike a liver biopsy. Studies have shown that MRI is accurate in measuring the LIC (8). One point that has emerged is the ability of the MRI to look at the liver in its entirety. This provides an advantage over a liver biopsy, which only looks at a small sample of the liver. It is believed that iron deposition is not homogeneous, and, therefore, depending on the biopsy can result in sampling error (7). We found extremely intriguing results when we compared LIC with fibrosis. There was no statistical difference among fibrosis scores of 0, 1, or 2 when comparing liver iron concentrations. LIC is an indirect measurement of liver injury. This important comparison demonstrates that although MRI may provide a statistically similar LIC, it does not provide clinicians with information about liver injury, which should guide the therapy. Because this 9-year retrospective review included time before the MRI assessment of liver iron, we did not evaluate the LIC obtained by MRI as it relates to liver fibrosis; however, because our results demonstrate that LIC and fibrosis do not correlate, MRI may not provide clinicians with the proper information to make decisions regarding therapy.

Some limitations of the study deserve mention. We acknowledge that there is a sampling error in liver biopsies given the fact that iron burden is not homogeneous. In addition, in a retrospective chart review, it was difficult to determine whether the ferritin levels were elevated as an acute-phase reactant or because of iron overload. We also recognize that the present study focused only on patients with sickle cell and did not include patients with thalassemia or those who required serial transfusions for other reasons. Finally, the present study did not take into account how long patients had been receiving serial transfusions and/or the rate of transfusions each patient had received at the time of biopsy. This additional piece of information can also assist clinicians while making therapy changes.

In conclusion, we describe the largest number, to date, of liver biopsies from patients with sickle cell evaluated in a single study for liver iron concentration, portal/periportal inflammation, and fibrosis. We demonstrated a statistically significant correlation between ferritin concentration and LIC, but this correlation does have variability (R2 = 0.55). NTBI may provide greater accuracy of organ damage but is only available in a few research centers. NTBI is unbound to transferrin and is toxic to organs. In the future, if NTBI becomes more accessible to clinicians, this test may replace ferritin for surveillance of iron risk. Previous studies have demonstrated a strong association between LIC and MRI iron. Our data suggest that, except for the highest level of fibrosis, there is no statistical association between LIC and liver fibrosis. These data highlight a major limitation of MRI that should be considered by clinicians when determining whether to perform a liver biopsy or MRI to assess iron-induced pathology of the liver. This article suggests that a combination of liver biopsy and MRI, thereby decreasing the frequency of each, may be the most beneficial in assisting the medical management of patients with sickle cell on chronic transfusions.

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REFERENCES

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18. Batts K, Ludwig J. Chronic hepatitis: an update on terminology and reporting. Am J Surg Pathol 1995; 19:1409–1417.

Keywords:

ferritin; fibrosis; liver iron content; sickle cell; transfusions

© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,

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