Background and aimsSampling size variability of liver biopsy remains a major limitation in the assessment of liver fibrosis. We aimed to evaluate the diagnostic value of a fully quantitative method (second harmonic generation/two-photon excitation fluorescence, SHG/TPEF based) in short liver biopsy samples.MethodsLiver biopsy samples from chronic hepatitis B (CHB) patients were constructed into virtual biopsies with different lengths. The original and virtual samples were measured by SHG/TPEF-based technology to obtain qFibrosis score, respectively. Here, Delta qFibrosis was defined as difference of qFibrosis between original biopsy and virtual biopsy. Equivalence test was used to compare Delta qFibrosis with the clinically acceptable error (deviation of 0.50) in each group.ResultsIn real-world practice, qFibrosis score increased significantly with fibrosis progression in >= 1.5-cm-, 1.0-1.5-cm-, and 0.5-1.0-cm-long specimens (p<0.05), compared with <= 0.5-cm-long specimens (p>0.05). In virtual biopsy samples with specified length, the equivalence was confirmed in 0.5-1.0-cm- and 1.0-1.5-cm-long specimens (0.27 vs. 0.22, p<0.001), whereas not in <= 0.5-cm-long specimens (0.53, p>0.05). The number of cross-linked collagen fibers, the total and aggregated collagen proportionate area, and the collagen strings in number, length, width and perimeter showed excellent consistency with original biopsy samples in 0.5-1.0-cm- and 1.0-1.5-cm-long specimens (ICC>0.90).ConclusionsThe use of SHG/TPEF-based image technology may give useful suggestive information in evaluation of CHB-related liver fibrosis for the short sample (biopsy length>0.5cm).