Determination of Hepatic Iron Concentration in Fresh and Paraffin-Embedded Tissue: Diagnostic Implications zyxwvutsrqponmlkjihgfedcbaZYXWVUTS JOHN K. OLYNYK, ROSEMARY O’NEILL, ROBERT S. BRITTON, and BRUCE R. BACON Division of Gastroenterology and Hepatology, Department of Internal Medicine, St. Louis University Health Sciences Center, St. Louis, Missouri Background/Aims: Determination of hepatic iron con- centration (HIC) is essential for the evaluation of hered- itary hemochromatosis. Occasionally, only paraffin-em- bedded liver biopsy specimens are available, or fresh biopsy specimens have been placed in saline for trans- port. This study aimed to describe a method for extrac- tion of liver tissue from paraffin blocks, determine the accuracy of measurement of HIC in recovered tissue compared with fresh tissue, and determine the effect of immersion in saline on HIC. Methods: HIC was mea- sured in both fresh and deparaffinized liver specimens (n = 41). Accurate measurements were defined as ei- ther a normal result in both specimens or a result in the deparaffinized specimen that was within 30% of the fresh measurement. Results: Measurements of HIC in fresh and deparafhnized tissue showed an excellent linear relationship (r = 0.95). In deparaffinized samples ~0.4 mg, accurate measurements were seen in 24 out of 29 specimens, compared with 6 out of 12 specimens weighing ~0.4 mg (P < 0.01). The hepatic iron index calculated from results in deparaffrnized samples ~0.4 mg correctly classified all patients. Immersion of fresh biopsy specimens in saline for 1 hour resulted in up to 50% iron loss (P < 0.05). Conclusions: Accurate measurement of HIC in deparaffinized liver biopsy spec- imens is possible. Calculation of the hepatic iron index from deparaffrnized liver tissue can facilitate diagnosis of hemochromatosis when fresh tissue is not available. Samples should not be transported in saline. iver biopsy for histopathology and biochemical mea- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED L surement of nonheme hepatic iron concentration (HIC) is essential for the accurate diagnosis of hereditary hemochromatosis (HHC).lm3 Furthermore, measurement of the HIC and calculation of the hepatic iron index (HII) (HIUage) enables discrimination between patients with homozygous HHC and those with either the hetero- zygous form of the disease or other secondary causes of hepatic iron deposition.‘,2,4-s Biochemical measurements of HIC are usually performed on fresh tissue submitted at the time of biopsy. In most instances, the liver biopsy specimen for measurement of HIC is submitted in a clean, dry container. Then, the specimen is dried, acid- digested, and the HIC is measured by either atomic ab- sorption spectrophotometry2’” or a calorimetric assay. lo However, on occasion, either fresh tissue is not available for measurement of HIC or the liver biopsy specimen has been transported in saline. When fresh tissue is not available, quantitative histological grading4,” and bio- chemical determination from paraffin-embedded tissue following deparaffinization’* have been suggested as methods for measuring HIC. However, to date, there have been no reports describing the methodology and/ or accuracy of measurement of HIC in liver specimens recovered from paraffin blocks compared with measure- ments in fresh tissue. In addition, although there have been reports supporting minimal loss of hepatic iron when biopsy specimens are placed in formalin,‘09’3 there have been no reports concerning the placement of liver biopsy specimens in physiological saline and the effect this might have on the accuracy of measurement of HIC when compared with fresh specimens. Accordingly, the aims of this study were to: (1) de- scribe a method for extraction of liver tissue from paraf- fin-embedded blocks and to determine the accuracy of measurement of HIC in recovered tissue compared with fresh tissue and (2) determine the accuracy of measure- ment of HIC in liver biopsy specimens following immer- sion in saline compared with fresh tissue. zyxwvutsrqponmlkjihgfedc Materials and Methods Human Biopsy Samples Liver biopsy specimens for histopathological examina- tion and biochemical measurement of HIC were obtained dur- ing routine clinical evaluation from 41 patients for the compar- ison of measurements of HIC in fresh and deparaffinized specimens (10 HHC, 1 transfusional iron overload, and 30 other various liver diseases). HHC was diagnosed on the basis of standard clinical, biochemical, and histopathologic assess- ment.’ At the time of biopsy, the sample was divided into Abbreviations used in this paper: HHC, hereditary hemochre matosis; HIC, hepatic iron concentration; till, hepatic iron index. 0 1994 by the American Gastroenterological Association 0016-5085/94/$3.00