Second Harmonic Generation Reveals Subtle Fibrosis Differences in Adult and Pediatric Nonalcoholic Fatty Liver Disease

Feng Liu, MD; Jing-Min Zhao, MD; Hui-Ying Rao, MD; Wei-Miao Yu, MD; Wei Zhang, MD; Neil D. Theise, MD; Aileen Wee, MBBS, FRCPath, FRCPA; and Lai Wei, MD

Disclosures

Am J Clin Pathol. 2017;148(6):502-512. 

In This Article

Results

Summary of Clinical Data

Clinical data are summarized in Table 1. Sixty-two adult patients, including 35 (56%) women, had a mean age of 43 years (range, 18–76 years). In all, 23 (37%) patients were overweight, and a further 21 (34%) patients were obese. Thirty-six pediatric cases, including six (13%) girls, had a mean age of 12 years (range, 6–17 years) and also were largely overweight (13 [36%] patients) or obese (20 [56%] patients). There were statistical differences among body mass index,[24,25] alanine transaminase, aspartate transaminase, γ-glutamyl transferase, alkaline phosphatase, and platelet count between adult and pediatric patients with NAFLD (P < .05).

Summary of Histologic Findings

Histologic staging of fibrosis in the study LBx is presented in Table 2. In LBx showing early stage NAFLD (stages 1A, 1B, 1C, and 2; ie, when fibrous septa have not distorted liver architecture and thereby obscured landmarks), most adult patients fell into category stage 1A among all stage 1 patients. Only a few patients exhibited stage 2 disease; that is, portal fibrosis was present in only five (26%) of 19 early fibrotic LBx and five (8%) of 62 adult specimens. For pediatric patients, most with early stage NAFLD had some degree of portal fibrosis (stages 1C and 2), comprising 16 (84%) of 19 early fibrotic LBx and 16 (44%) of 36 pediatric specimens Figure 1. According to NASH CRN assessment of patterns of steatosis, in the pediatric group, three patients showed zone 3 steatosis, one patient showed zone 1 steatosis, and the remainder were panacinar.

Figure 1.

Portal vs central fibrosis in early stage adult and pediatric nonalcoholic fatty liver disease. Most adults had stage 1A fibrosis (ie, predominantly central) while most children displayed stage 1C and 2 disease (ie, predominantly portal, with or without central fibrosis).

qFibrosis Assessments

Liver fibrosis progression was quantified using the SHG/TPEF system by examining the collagen levels and morphology. We first investigated the performance of qFibrosis to replicate the fibrosis scores obtained with the NASH CRN system. Patterns of collagen detected by SHG matched the light microscopic appearances and histochemically stained collagen patterns with high fidelity Image 1. Based on their quantitative trends with respect to the fibrosis stages and systemic AUC analyses, six shared parameters mainly for string collagen (StrLength, StrWidth, StrEccentricity, StrSolidity, #StrPT, and #ShortStrPT) were selected from both patient groups; they were demonstrated to identify differences among all fibrosis stages with high AUC (AUC: 0835–0.982 in the adult group, 0.885–0.981 in the pediatric group) Table 3.

Image 1.

Comparison of histopathologic staining (H&E and Masson trichrome) with second harmonic generation (SHG)/two-photon excitation fluorescence images of liver biopsy tissue from (A) adult patients with nonalcoholic fatty liver disease (NAFLD). (B) pediatric patients with NAFLD. SHG imaging, Masson trichrome, and H&E were performed on serial sections (×200).

Quantitative Features in Portal Tract Collagen Between Adult and Pediatric Groups

The quantitative features in the portal region exhibited similar trends when adult and pediatric LBx were compared, representative highlights of which are shown in Figure 2A and Figure 2B (the remaining data in the supplementary materials). As expected, we found that normal adult PTs showed baseline normal stroma that was greater than normal pediatric PTs, although the differences did not reach statistical significance. All collagen parameters in the portal region showed similar patterns of change when stages 0, 1C, and 2 were compared in both patient groups. In pediatric patients, the portal qFibrosis parameters remained largely unchanged between stages 1C and 2, with the difference between these two stages appearing to be merely the addition of zone 3 collagen without a change in the PTs. In adults, however, the change from stage 1C to 2 seemed to involve a marked increase in PT stroma.

Figure 2.

Comparison between adult and pediatric nonalcoholic fatty liver disease of (A) the number of collagen intersections in the portal tract region, (B) the percentage of aggregated collagen in the portal tract region, (C) the number of aggregated long collagen strings in the central vein region, and (D) the number of distributed thick collagen strings in the central vein region. *P < .05. **P > .05.

Quantitative Features in the CV Collagen Between Adult and Pediatric Groups

Selected representative data concerning changes in CV regions in early stage fibrosis for both patient groups are shown in Figure 2C and Figure 2D (full data presented in the supplementary materials). Adults have higher normal baseline collagen percentage area around CV regions compared with pediatric patients, similar to collagen percentage area around PT regions, but again, not statistically significant. In CV regions, two patterns of change were discernible, rather than the single pattern seen for PT. First, we observed that all parameter values decreased in adults when progressing from stage 1A/B to 2, but in pediatric cases, nearly all aggregated collagen parameters (Figure 2C) decreased and nearly all distributed collagen parameters (Figure 2D) increased from stage 1A/B to 2.

Quantitative Features in the Advanced Stages Between Adult and Pediatric Groups

In advanced stages (3 and 4, combined), selected representative data are shown in Figure 3 (remainder of data presented in the supplementary materials). Parameters in CV regions were markedly increased in pediatric cases from normal baseline, while adult CV parameters (including size by percent area and all collagen parameters) were largely unchanged (Figure 3A). In PT regions, we observed highly significant increases in PT size (by percent area) and in all collagen parameters for both adult and pediatric groups (Figure 3B). There were no statistical differences for collagen parameter in CV regions and PT regions in the advanced stages between adult and pediatric groups.

Figure 3.

Comparison between adult and pediatric nonalcoholic fatty liver disease of (A) the number of collagen intersections in the central vein region and (B) the number of collagen intersections in the portal tract region. *P < .05. **P < .001. ***P > .05.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....