Costaining for keratins 8/18 plus ubiquitin improves detection of hepatocyte injury in nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease is a global health dilemma. The gold standard for diagnosis is liver biopsy. Ballooned hepatocytes are histologic manifestations of hepatocellular injury and are characteristic of steatohepatitis, the more severe form of nonalcoholic fatty liver disease. Definitive histologic identification of ballooned hepatocytes on routine stains, however, can be difficult. Immunohistochemical evidence for loss of the normal hepatocytic keratin 8/18 can serve as an objective marker of ballooned hepatocytes. We sought to explore the utility of a keratin 8/18 plus ubiquitin double immunohistochemical stain for the histologic evaluation of adult nonalcoholic fatty liver disease. Double immunohistochemical staining for keratin 8/18 and ubiquitin was analyzed using 40 adult human nonalcoholic fatty liver disease core liver biopsies. Ballooned hepatocytes lack keratin 8/18 staining as previously shown by others, but normal-size hepatocytes with keratin loss are approximately 5 times greater in number than keratin-negative ballooned hepatocytes. Keratin-negative ballooned hepatocytes, normal-size hepatocytes with keratin loss, and ubiquitin deposits show a zonal distribution, are positively associated with each other, and are frequently found adjacent to or intermixed with fibrous matrix. All 3 lesions correlate with fibrosis stage and the hematoxylin and eosin diagnosis of steatohepatitis (all P < .05). Compared with hematoxylin and eosin staining, immunohistochemical staining improves the receiver operating characteristics curve for advanced fibrosis (0.77 versus 0.83, 0.89, and 0.89 for keratin-negative ballooned hepatocytes, normal-size hepatocytes with keratin loss, and ubiquitin, respectively) because immunohistochemistry is more sensitive and specific for fibrogenic hepatocellular injury than hematoxylin and eosin staining. Keratin 8/18 plus ubiquitin double immunohistochemical stain improves detection of hepatocyte injury in nonalcoholic fatty liver disease. Thus, it may help differentiate nonalcoholic steatohepatitis from nonalcoholic fatty liver.

FIGURE 1

H&E and IHC stained sections showing hepatocellular injury. (A) An H&E stained section shows easily identified enlarged, rounded, swollen-appearing ballooned hepatocytes (arrow heads) with cytoplasmic clearing and reticulation and perinuclear Mallory-Denk bodies. (B) A Masson trichrome stained section shows numerous ballooned hepatocytes (arrow heads) with associated fibrosis. (C) K8/18 plus ubiquitin double IHC stained section shows several enlarged keratin negative ballooned hepatocytes (KBH) (arrow heads). The red-chromagen tagged ubiquitin antibody highlights Mallory-Denk bodies. The adjacent normal size hepatocytes show the usual homogeneous cytoplasmic staining with the brown chromagen-tagged K8/18 antibody. (D) Many normal size hepatocytes show complete loss of K8/18 staining (KH) (arrows). These KH also contain Mallory-Denk bodies. (E and F) Typical examples of foci of hepatocellular injury as seen with the double IHC stain. KBH (arrow heads) and KH (arrows) are often co-localized and adjacent to or intermixed with fibrosis. Many ubiquitinated protein aggregates are perinuclear, however, extracellular ubiquitin deposits (asterisks) can often also be identified. (G) In some cases, K8/18 staining was faint and patchy, however, KBH (arrow heads) and KH (arrows) can still be identified. (H1–H2) H1shows an H&E stained section of a somewhat linear area of fibrosis in which ballooned hepatocytes are not detected. H2 shows the double IHC stained section of the same focus; hepatocytes with loss of keratin staining and ubiquitin deposits are easily seen.

FIGURE 2

Contingency plot analyses comparing H&E scoring parameters with IHC detection of hepatocellular injury. (A) The H&E scoring of ballooned hepatocytes is compared to the IHC analysis of KBH, (B) the H&E scoring of MDBs is compared to the IHC analysis of Ub deposits, (C) the H&E diagnosis of SH is compared to the IHC analysis of KBH, (D) the H&E diagnosis of SH is compared to the IHC analysis of Ub deposits, and (E) the H&E diagnosis of SH is compared to the IHC analysis of KH.

FIGURE 3

Box plot graph with HOMA-IR (mg/dl*µU/ml) shown on the vertical axis and ‘None’ (0 per 10 HPFs), ‘Few’ (>0 and <10 per hpfs) and ‘Many’ (≥10 per hpfs) ubiquitin particles, KBH, and KH on the horizontal axis. The boxes represent the median (middle line), 25^th percentile (bottom line of the box), and 75^th percentile (top line of the box). The lowest and highest datum within 1.5 inter-quartile ranges for each category is represented by the ends of the whiskers. Median values of ‘None’, ‘Few’ and ‘Many’ with p-values were 3.8, 5.8, and 7.6 for ubiquitin particles (p=0.08), 3.6, 9.2, and 8.4 for KBH (p=0.02), and 3.8, 6.5, and 7.6(p=0.15) for KH.