Reference method for measurement of the hepatic iron concentration
Authors: Gary M. Brittenham
Journal: American Journal of Hematology (2014)

What should be the reference method for measurement of the hepatic iron concentration? In this issue of the American Journal of Hematology, Urru and colleagues 1 present further evidence that, in patients with thalassemia major and transfusional iron overload, chemical measurement of the iron concentration in a desiccated liver-biopsy specimen obtained by percutaneous biopsy is highly reproducible, as judged by measurement in a successive specimen, provided that two conditions are met. The first condition is the absence of cirrhosis, defined in this study as Ishak scores 2 of 5 or 6, because, with cirrhosis, the iron distribution within the liver is no longer uniform. The second condition is that the liver-biopsy specimen is adequate, ≥1 mg dry weight; variability increases with smaller samples. No complications were observed with liver biopsy, extending the record of safety of liver biopsy in expert hands 3-5. These results corroborate previous observations from these and other investigators 5-7 that support chemical measurement of the iron concentration in a liver-biopsy specimen as the reference standard for measurement of the hepatic iron concentration. Still, dissonance in the details must be acknowledged; no standard method for liver biopsy procedure, specimen processing and analysis has been established. The biopsy needle (cutting needle or Menghini needle with saline flushing), exposure to saline, formalin, and fixation solutions, use of iron-free containers, specimen processing (fresh or paraffin-embedded; methods of deparaffinization and of digestion), methods of analysis (colorimetric, atomic absorption spectroscopy, inductively coupled plasma mass spectroscopy), and other factors differ among published studies and potentially influence results 5, 6. Hepatic iron concentrations measured in deparaffinized samples were a mean of 23% greater than those in desiccated tissue from fresh liver specimens 6. The minimum weight for an adequate liver sample for analysis remains uncertain; evidence has been provided both for thresholds of 0.4 mg dry weight 8, 9 and for 1 mg dry weight 1, 4, 5. Reports of variability in the hepatic iron concentration in different areas of the liver have generally been restricted to studies of cirrhotic livers 10-12; data are lacking in patients with transfusional iron overload without cirrhosis. Even so, the close relationship observed between the hepatic iron concentration and total body iron stores 4 in transfused patients with thalassemia who were free of cirrhosis suggests that the extent of variability is limited. An alternative view of the reference method for measurement of the hepatic iron concentration is that magnetic resonance imaging (MRI), recommended in a variety of guidelines, has become the de facto technique. Nonetheless, since acceptance of the manuscript by Urru et al. 1 for publication, a painstaking systematic review and meta-analysis of this use of MRI has appeared, 13 concluding that “measurements of liver iron concentration by MRI may be accurate enough to rule out iron overload, but not to definitely identify patients with this condition.” An accompanying editorial concurs with this assessment and provides some helpful guidance for clinicians 14. The systematic review emphasized the low quality of existing studies “both at the conduct and reporting levels” and the paucity of available evidence 13, even for the protocol approved by the U.S. Food and Drug Administration (FerriScan; Resonance Health, Claremont, Australia). Other recent commentaries have emphasized that studies using T2, R2, T2*, and R2* MRI parameters typically have been calibrated against chemical measurements of hepatic iron in biopsy samples by using only a single scanner and cautioned that such calibrations are not transferable 15. Depending on the calibration used, a liver T2* of 2.5 ms measured on a 1.5-T scanner corresponds to a hepatic iron concentration ranging from 5.4 mg Fe/g dry weight 16 (<7.0 mg Fe/g dry weight; low risk of complications) 17 to 26.4 mg Fe/g dry weight 18 (>15.0 mg Fe/g dry weight; high risk) 15. This example is consistent with the experience of clinicians who may receive widely varying estimates of the hepatic iron concentration in the same patient evaluated contemporaneously by different MRI scanners. In addition to considerations of calibration, MRI detects tissue iron indirectly by the effects of the storage (nonheme) forms of iron on hydrogen nuclei. The two forms of storage iron, ferritin and hemosiderin, have distinctively different effects on MRI signal decay 19. Methods to measure the total storage (ferritin + hemosiderin) iron based on a single metric (T2, R2, T2*, and R2*) have an intrinsic uncertainty resulting from variations in the relative amounts of ferritin and hemosiderin iron. Other factors, such as the presence of fat in the liver, may also confound MRI estimates of the hepatic iron concentration 20. Altogether, the present data uphold chemical measurement of the iron concentration in a liver-biopsy specimen obtained by percutaneous biopsy as the optimal reference method. Nonetheless, a critical role for biopsy will be to calibrate candidate MRI techniques for measurement of liver iron. To serve as a trustworthy standard, a uniform protocol for liver biopsy procedure, specimen processing and analysis is needed to permit meaningful interlaboratory comparisons 6. The results from Urru and associates 1 in this issue of the Journal indicate that such a protocol is feasible. Inevitably, MRI will increasingly be used for clinical purposes but, as the systematic review 13 and editorial 14 emphasize, much more work is needed to provide consistent, clinically reliable results.