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. 2015 Mar 11;6(4):1209-18.
doi: 10.1364/BOE.6.001209. eCollection 2015 Apr 1.

Evaluation of Area-Based Collagen Scoring by Nonlinear Microscopy in Chronic Hepatitis C-induced Liver Fibrosis

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Free PMC article

Evaluation of Area-Based Collagen Scoring by Nonlinear Microscopy in Chronic Hepatitis C-induced Liver Fibrosis

David Sevrain et al. Biomed Opt Express. .
Free PMC article

Abstract

In this paper we analyze a fibrosis scoring method based on measurement of the fibrillar collagen area from second harmonic generation (SHG) microscopy images of unstained histological slices from human liver biopsies. The study is conducted on a cohort of one hundred chronic hepatitis C patients with intermediate to strong Metavir and Ishak stages of liver fibrosis. We highlight a key parameter of our scoring method to discriminate between high and low fibrosis stages. Moreover, according to the intensity histograms of the SHG images and simple mathematical arguments, we show that our area-based method is equivalent to an intensity-based method, despite saturation of the images. Finally we propose an improvement of our scoring method using very simple image processing tools.

Keywords: (110.2960) Image analysis; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine; (180.4315) Nonlinear microscopy.

Figures

Fig. 1
Fig. 1
Representative SHG images of low (a,c) and high (b,d) fibrosis groups. (a) and (b) are raw SHG images. The PMT supplied voltage was adjusted so as to obtain a mean reference intensity of 4% of the full intensity range in a non-fibrotic area of (a). The corresponding raw TPEF images are shown in inset and are used to define a mask that delimits the contour of the biopsy and is applied to SHG images. (c) and (d) are binarized SHG images after 25% thresholding.
Fig. 2
Fig. 2
SHG index versus Metavir (a) and Ishak (b) scores. The box chart graph is defined as follow: the whiskers display extreme values whereas the boxes represent the median, 25th and 75th percentiles. The mean values are represented by the small box (▫). The numbers between brackets give the number of samples in each histological stage.
Fig. 3
Fig. 3
ROC curves of SHG index for the detection of advanced fibrosis for (a) Metavir (F012 vs F34) and (b) Ishak (I0123 vs I456). The area under the curve is 0.84 (95% confidence interval: 0.77-0.92) for Metavir and 0.82 (95% confidence interval: 0.74-0.91) for Ishak.
Fig. 4
Fig. 4
(a) Comparison of the SHG index of 35 samples obtained for two different detection gains (106 versus 105V/A) of the microscope but identical threshold-to-baseline ratios. The straight line is the best linear fit (slope = 0,98, R2 = 0,995). (b) Comparison of the percentages of the saturated pixels of SHG images of the same 35 samples for the two different detection gains (106 versus 105V/A).
Fig. 5
Fig. 5
Normalized SHG index ratio averaged on 3 pairs of samples with extreme fibrosis status (F4 to F1-F2 ratio) versus the value of the threshold used for calculating the SHG index.
Fig. 6
Fig. 6
(a) Total SHG intensity of the thresholded pixels per unit area of the samples and (b) mean SHG intensity per thresholded pixel as a function of the SHG index.
Fig. 7
Fig. 7
SHG intensity histograms of two biopsies with low (box) and high (triangle) fibrosis status. The inset displays the relevant part of these two histograms in semi-logarithmic scale in order to evidence the almost exponential behavior. The bin width of the histograms was set to 10 in order to reduce fluctuations.
Fig. 8
Fig. 8
SHG binary image before (a) and after (b) median filtering for a patient with F4-I6 histological status. The inset is a zoom on a non fibrotic area and demonstrates that isolated collagen deposits is removed by median filtering.
Fig. 9
Fig. 9
ROC curves of SHG index for the detection of advanced fibrosis for (a) Metavir (F012 vs F34) and (b) Ishak (I0123 vs I456), after median filtering of the binarized SHG images. The area under the curve is 0.87 (95% confidence interval: 0.79-0.94) for Metavir and 0.85 (95% confidence interval: 0.77-0.93) for Ishak.

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