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Discussion
Liver fibrosis is a major parameter guiding the diagnosis and prognosis of chronic liver disease. Studies based on liver biopsies to evaluate fibrosis in chronic hepatitis C usually relies on categorical scoring systems rather than direct measurement of the amount of fibrous tissue. Several DIA techniques for liver fibrosis quantification have been developed. It is considered as a promising tool because it provides results on a continuous scale, rather than merely five or seven qualitative stages. In this article, we described an inexpensive and worldwide available DIA technique that provides objective quantification of fibrosis in liver biopsies without the need of expensive digital glass-scanning devices and third party software.
Adobe® Photoshop® is largely used in the biomedical literature and considered 'inexpensive and commonly available' imaging software. Two previous studies described DIA techniques for liver fibrosis quantification using older versions of this software. The newer versions can be easily acquired from the World Wide Web. This software upgrade combined with the improvement of hardware performance allowed the development of very powerful image analysis tools. These make possible the whole sample digitalization (virtual large field images of the biopsies) without needing expensive glass-scanning devices.
Although Sirius Red is known as one of the best techniques for collagen histochemistry quantification of liver fibrosis, the authors preferred to use Masson's trichrome stain. This staining method is worldwide available in most pathology laboratories, and thus, more suitable to the purposes of the present study. A perfect contrast between fibrous tissue stained in blue and parenchyma stained in red was obtained following the 'Selective color' procedure described in the methodology, using the Masson's trichrome stain. We reinforce that similar results can be reached in a liver biopsy slide stained with Sirius Red by adjusting different colour pallets: reds and yellows. In samples stained by this histochemical method, red and yellow colours will be representative of fibrous tissue and parenchyma, respectively.
Our DIA technique had an excellent correlation with the traditional semiquantitative Ishak score and METAVIR classification (r = 0.95 and r = 0.92, respectively). Other studies have already observed a high correlation between DIA techniques and staging. It demonstrated a high capacity of DIA techniques in discriminating the different stages of fibrosis. Our study includes a larger sample and evaluated a cheaper method. Another advantage of the DIA method described in this study is its reproducibility with high rates of intra-observer concordance. We showed an intraclass correlation index of 0.99 (95% CI 0.95–0.99). This reinforces its reliability. Further studies are welcome to define interobserver variability and intercenter reproducibility. A small amount of training time and minimal knowledge in informatics are enough to accurately reproduce the method described. We anticipate that an easy learning curve, low cost and use of worldwide available technology will permit the application of this method in most pathology laboratories.
Important clinical cut-off points for septal fibrosis and for cirrhosis were established in this study. Patients with fibrosis index higher than 6% would have indication for antiviral therapy, while patients with fibrosis index higher than 27% would have indication for endoscopy and hepatocellular carcinoma surveillance. The role of these cut-off points in clinical practice should be better investigated in further studies.
The strengths of our DIA method were that images were processed on colour RGB model, the area of fibrosis was not manually manipulated, the software has adjustments of colours allowing a greater contrast between fibrosis and normal hepatic tissue, and the hardware/software packet is inexpensive and worldwide available. It also should be highlighted that all the fibrosis stages were well represented in this large sample size. Thus, this DIA method can be apply in current clinical practice with a very low cost.
In our methodology, the quantification of fibrosis automatically selects all blue sample allowing to map fibrous tissue including the delicate perisinusoidal fibrosis, which is usually undetected by examiner's eye. The precisely quantification of perisinusoidal fibrosis might be an advantage of this method in quantitative assessment of liver fibrosis in patients with others chronic liver disease than CHC, especially nonalcoholic fatty liver disease (NAFLD). Digital quantification of fibrosis by this method can be a future tool to ease the differentiation between patients with simple steatosis from those with nonalcoholic steatohepatitis (NASH). It can also contribute for a more precise staging in the last condition.
The histological diagnosis based on semiquantitative architectural changes in association with DIA techniques may express more precisely the actual fibrosis state. This combination allows evaluating not only the fibrosis distribution but also the fibrosis amount. We agree with the idea that DIA techniques are not suitable for replacing traditional qualitative and semiquantitative liver biopsy evaluation. More important, it should be considered as a complementary tool for the traditional histological methods.
Some potential applications for DIA techniques as complementary tool could be expected. In patients undergoing sequential liver biopsies showing the same stage, the variation in fibrosis index could estimate more precisely the progression of fibrosis. Another potential application could be in clinical trials for measuring the effect of novel antifibrogenic therapies when more objective and broader scale information is needed. This would be important for the validation of new noninvasive imaging techniques and indirect serum markers of fibrosis. It could also be useful in case of discordance of staging among different pathologists.
In summary, the DIA technique for liver fibrosis quantification described in this article is inexpensive and was developed using worldwide available technologies. The process is simple, reproducible and showed a high correlation with semiquantitative scores. It provides a more complete evaluation of fibrosis adding the quantification to the architectural patterns.
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