Phosphotungstic Acid–treated Picrosirius Red Staining Improves Whole-slide Quantitative Analysis of Collagen in Histological Specimens

Abstract

We tried to prevent nonspecific nuclear staining (NS-NS) of picrosirius red (PSR) staining by treating the specimens with one of the heteropoly acids phosphotungstic acid (PTA). We analyzed a total of 35 cases of non-cancerous liver tissue for fibrosis and NS-NS under PSR-alone, phosphomolybdic acid (PMA)-pretreated PSR (PMA + PSR), or PTA-pretreated PSR (PTA + PSR) condition. In addition, we analyzed the photosensitivity of PMA or PTA single stain specimens. PTA + PSR significantly suppressed NS-NS compared with PSR. The color of the specimens did not change into blue by 30 times the exposure to whole slide scanner (WSS) light. The PTA + PSR condition showed the highest correlation with the Ishak score (pathological evaluation of liver fibrosis) compared with other conditions. Furthermore, Sirius Red–positive percentage (SRP%) in PSR was increased in the NS-NS observed cases. SRP% in PMA + PSR was significantly affected by WSS light exposure time. Moreover, the deposition of non-polarized PSR-stained substances (NP-PSR⁺S) clinging to the collagen fibers potentially explains why SRP% seemed bigger under PSR than PTA + PSR. Our protocol enabled us to analyze the whole slide image of PSR staining by high magnification, which would contribute to the accurate analysis of collagen amount in the tissue sections.

Introduction

Chronic liver disease is caused by various factors, including hepatitis B or C virus infection, alcohol abuse, biliary obstruction ¹ helminth infection, and hereditary metal overload, ² resulting in hepatic fibrosis development. The degree of liver fibrosis plays a vital role in diagnosing and assessing the prognosis for chronic liver diseases. ³ In the evaluation of liver fibrosis, specific stains, such as Mallory, Masson’s trichrome, Sirius Red, Elastica van Gieson, ⁴ Orcein staining, ⁵ and immunohistochemistry for each collagen type, ⁶ are used. Moreover, Sirius Red F3BA, used for Sirius Red staining, is a very stable dye and stains all collagen fibers red and is suitable for polarized observation. ⁷ In addition, Sirius Red staining is a selective dye for collagen fibers ⁸ and has been established as the most stable method of visualizing fibrosis in liver biopsies. ⁹ Computer-assisted image analysis (CAIA), as well as Masson’s trichrome, ^{10 -12} reticulin, ¹³ Sirius Red,^14-16 and Elastica van Gieson staining,^17-19 is used widely for liver fibrosis assessment.

Furthermore, liver fibrosis analysis was performed using whole slide scanners (WSSs) recently. For example, we use WS data in the research area to measure collagen proportionate area (CPA) using a digital image analysis software for liver biopsy tissues. ¹⁴ We previously reported that phosphomolybdic acid (PMA) pretreatment is effective in overcoming the nonspecific nuclear staining (NS) of Sirius Red staining to adapt Sirius Red staining for CAIA. ²⁰ However, our method was difficult to use for WS data because of the formation of molybdenum blue by WSS light exposure. ²⁰ Thus, developing specific stains restraining the NS for WS data and preventing the formation of blue color by WSS light exposure is necessary for CAIA assessment. Usually, we use phosphotungstic acid (PTA) or PMA in Masson’s trichrome and Mallory’s trichrome staining as mordant in each procedure. ²¹ In addition, both PMA and PTA are categorized as heteropoly acids.^22,23 Thus, we assumed that not only PMA pretreatment but also PTA pretreatment could block nonspecific NS in Sirius Red staining. Therefore, this study aimed to examine the difference in photosensitivity between PTA and PMA on tissue specimens and the use of PTA pretreatment in slide scanning by a WSS.

Methods

Samples of Human Tissues

To compare PMA and PTA pretreatment in picrosirius red (PSR) staining, we used 17 cases of non-cancerous tissue of primary liver cancer (15 males and 2 females; mean age, 73.8 years) and 18 cases of non-cancerous tissue of metastatic liver cancer (15 males and 3 females; mean age, 66.3 years), which were prepared in routine clinical practice in Gunma University Hospital between January 1, 2005, and December 31, 2020. In routine clinical practice, the tissues were fixed using 10% formaldehyde in phosphate-buffered saline (PBS) for 48 hr, and paraffin blocks were prepared. To determine the state of fibrosis in 17 cases of non-cancerous tissue of primary liver cancer, two pathologists (M.S. and H.I.) independently estimated fibrosis based on Ishak score. ²⁴ If the score differed between two pathologists, another pathologist (H.Y.) estimated the specimens and the three pathologists discussed the estimation and determined fibrosis score. While examining the photosensitivity of PTA, we used newly prepared paraffin blocks from remaining liver tissue fixed in 10% formaldehyde for 6 months as extra samples after final diagnosis. We used five cases of non-cancerous tissue primary liver cancer (five males; mean age, 73.8 years) for the ultraviolet (UV) experiments and WSS light experiments discussed below.

This study was conducted after obtaining approval from the Gunma University Ethics Review Committee of Medical Research.

Sample of Mouse

We euthanized the Mdr2 gene knockout mouse at the age between 8 weeks and 10 weeks using cervical dislocation. Following the removal of the liver, it was fixed with 10% formaldehyde neutral buffer solution pH 6.9–7.1 (37152-51; Nacalai Tesque Inc., Kyoto, Japan) overnight. Then, we embedded tissues in paraffin using an auto tissue processor.

Four-µm-thick sections were prepared from paraffin blocks and used to study UV exposure experiments or WSS light exposure experiments, which follow the same procedure as those used for human samples.

Picrosirius Red Staining

We used Sirius Red (33061; Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and saturated picric acid (8766-1; Muto Pure Chemicals Co., Ltd.) to prepare PSR solution. In addition to PSR staining without mordant, we examined protocols using PMA (12 molybdo(IV) phosphoric acid n-hydrate; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) pretreatment or PTA (5% PTA solution, 4004-1; Muto Pure Chemicals Co., Ltd.) pretreatment as mordant. The protocol using PSR staining without mordant was called PSR staining in this study. The protocols with PMA pretreatment as mordant were called PMA + PSR staining, and the one with PTA pretreatment was called PTA + PSR staining in this study. These protocols are summarized in Table 1.

Table 1.

Protocols Used for Picrosirius Red Staining in the Present Study.

PSR	Times
Xylene (3 times)	5 min each
100%, 99%, 95%, 70% ethanol	1 min each
Running tap water	1 min
Rinse with distilled water	3 times
PSR solution	60 min
0.1 N hydrochloric acid (2 times)	5 min each
100% ethanol (3 times)	5 min each
Xylene (3 times)	5 min each
PMA + PSR	Times
Xylene (3 times)	5 min each
100%, 99%, 95%, and 70% ethanol	1 min each
Running tap water	1 min
0.2% PMA	2 min
Rinse with distilled water	3 times
PSR solution	60 min
0.1 N hydrochloric acid (2 times)	5 min each
100% ethanol (3 times)	5 min each
Xylene (3 times)	5 min each
PTA + PSR	Times
Xylene (3 times)	5 min each
100%, 99%, 95%, 70% ethanol	1 min each
Running tap water	1 min
0.2% PTA	2 min
Rinse with distilled water	3 times
PSR solution	60 min
0.1 N hydrochloric acid (2 times)	5 min each
100% ethanol (3 times)	5 min each
Xylene (3 times)	5 min each

Abbreviations: PSR, picrosirius red; PMA, phosphomolybdic acid; PTA, phosphotungstic acid.

Effects of Staining by Heteropoly Acid After Exposure to UV

Four-µm-thick sections were prepared, and the sections were soaked in xylene for 5 min 3 times and soaked in a series of alcohol solutions (100%, 99%, 95%, and 70% ethanol) for 1 min each. After rinsing with running water for 1 min, sections were soaked in 0.2% PMA or 0.2% PTA solution for 2 min. We rinsed sections with distilled water (DW) 3 times (moving up and down in the container 10 times) and immersed them in 100% ethanol twice for 1 min and then in xylene for 5 min 3 times. Afterward, we used the specimens for observation after placing the coverslip. The specimens were placed on the UV irradiation equipment (UV Transilluminator MD-20, 312 nm; input, 100 V, 50–60 Hz; power, max 50 W; fuse, 3.15 A, 250 V; dimensions [L × W × H], 321 × 281 × 119 nm; Wealtec Corporation, Sparks, NV) with the specimen side facing downward, and the specimens were exposed to the 312-nm 50-W UV for 5 min, covering the equipment using a shielding box. In addition, to prevent UV effects, we used UV-cut film (RT05HDSL; UV-A and UV-B, 99.5% cut; LINTEC, Tokyo, Japan) to cover the slides. To evaluate the color of specimens after UV exposure, we placed the specimens in a 20-cm light-emitting diode (LED) light–attached box with white paper in the background (PULUZ 20 cm LED Portable Photo Studio; Shenzhen Puluz Technology Limited, Longgang, Shenzhen, China). Subsequently, we photographed the whole slide using a digital camera attached with an 18–55 mm lens (Canon EOS Kiss X90; Canon, Tokyo, Japan) under the following conditions: lens, 55 mm; AV mode; auto white balance (AWB); ISO 200; aperture, 5.6; saving format, JPEG 4000 × 6000 pixel 72 dpi.

For tone evaluation, we analyzed JPEG images using Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA). First, we corrected the tone by setting the part of white background except the surface of the slide in photo images as white range and converted the images into grayscale. After surrounding the areas of specimens with an annotation tool, we measured the grayscale value in those areas and saved the average grayscale as Microsoft Excel data.

Scanning and Saving Specimen Images Using a Whole Slide Scanner

We scanned the stained specimens by WSS (Nano Zoomer-SQ, C13140-01; Hamamatsu Photonics K.K., Shizuoka, Japan) under the following conditions: objective lens, ×20 NA = 0.7; scan mode, ×40 mode; max capture size, 26 × 76 mm; 0.23 µm/pixel; illuminant LED; saving image format, JPEG; manual focus mode.

Analysis of TIFF Files of High-magnification Images

The scanned images were displayed at ×40 magnification using dedicated browsing software (NDP view 2, U12388-01; Hamamatsu Photonics K.K.). Subsequently, we selected part of liver lobules not containing Glisson’s sheath and recorded five random images per case. The information on the image of the area taken was saved using the annotation function of the software. The images were captured at these conditions: magnification, ×40; resolution, 300 dpi; image format, TIFF file (1920 × 1088 pixels). The TIFF images were converted to a TIFF file without compression using image conversion software (Irfan View Version 4.23; Irfan Skiljan, Wiener Neustadt, Austria). Then, the uncompressed TIFF images were converted to MRXS files using image conversion software (Slide Converter 1.13; 3DHISTECH Ltd., Budapest, Hungary) before being analyzed by image analysis software Histoquant (Version 2.1.1, Quant center module; 3DHISTECH Ltd.). For image analysis by Histoquant software, the following conditions were used for analysis: setting value—PSR except for the NS: no colocalization; noise reduction blur (Gauss) 0; hue–saturation–value (HSV) ranges: hue 348, 17; saturation 26, 40; separation 0; no fill holes; no filter. PSR that detects as maximum as possible collagen fiber areas regardless of the NS: noise reduction blur (Gauss) 0, ranges: hue 348, 17; saturation 15, 40; separation 0; no fill holes; no filter. PTA + PSR that detects as maximum as possible collagen fiber areas: noise reduction blur (Gauss) 0; ranges: hue 328, 9; saturation 11, 40; separation 0; no fill holes; no filter.

We reserved the percentage of Sirius Red–positive areas in the whole slide as Excel data.

Analysis of the Area of Fibrosis Using WS Data

We converted WS data (ndp file) into MRXS file using Slide Converter 1.13 (3DHISTECH Ltd.). Then we analyzed Sirius Red–positive areas in tissue sections under the following conditions using the Quant center module of Histoquant (3DHISTECH Ltd.). For murine liver analysis, we collected data on the percentage of Sirius Red–positive regions in the whole slide area and the whole liver area in the whole slide area and recorded them in Microsoft Excel. The percentage of the area of liver fibrosis was calculated by dividing the percentage of Sirius Red–positive regions by the whole liver area. The following conditions were used for analysis: setting value for Sirius Red–positive area—PSR that detects maximum collagen fiber areas as much as possible regardless of the NS: noise reduction blur (Gauss) 0; ranges: hue 348, 17; saturation 23, 33; separation 0; no fill holes; no filter. PTA + PSR that detects as maximum as possible collagen fiber areas: noise reduction blur (Gauss) 0; ranges: hue, 328, 9; saturation 22, 65; separation 0; no fill holes; no filter. PMA + PSR that detects the maximum possible collagen fiber areas: noise reduction blur (Gauss) 0; ranges: hue 324, 5; saturation 34, 77; separation 0; no fill holes; no filter. The setting values for the whole liver area for PSR were as follows: noise reduction blur (Gauss) 0; ranges: hue 336, 87; saturation 7, 55; separation 0; no fill holes; no filter. The setting values for the whole liver area for PMA + PSR and PTA + PSR were as follows: noise reduction blur (Gauss) 0; ranges: hue 332, 87; saturation 6, 65; separation 0; no fill holes; no filter.

For human liver specimens, we converted WS data (NDP files) into MRXS files using Slide Converter 1.13 (3DHISTECH Ltd.). Given that the capacity of data analysis of the computer used was small, we selected a section of the liver specimen for WS data analysis. We selected the same area by annotation function using Histoquant software for each staining method for the same cases. The percentage of Sirius Red–positive area in the annotation area was analyzed using the Quant center module in Histoquant software, and the data were recorded in an Excel file. The analysis conditions for each staining condition are listed subsequently. The settings for PSR specimen staining that detects the maximum possible collagen fiber areas regardless of the NS were as follows: noise reduction blur (Gauss) 0; ranges: hue 348, 7; saturation 17, 56; separation 0; no fill holes; no filter. The settings for PTA + PSR that detects the possible maximum collagen fiber areas were as follows: noise reduction blur (Gauss) 0; ranges: hue 323, 5; saturation 20, 67; separation 0; no fill holes; no filter. For PMA + PSR specimen staining, we prepared two settings. Setting 1 was for detecting the maximum possible Sirius Red–positive area of collagen fibers in the initial WS data: noise reduction blur (Gauss) 0; ranges: hue 324, 5; saturation 34, 77; separation 0; no fill holes; no filter. Setting 2 was for detecting the maximum possible Sirius Red–positive area of collagen fibers in the data after multiple scanning (at least 5 times): noise reduction blur (Gauss) 0; ranges: hue 328, 5; saturation 36, 85; separation 0; no fill holes; no filter. We collected the percentage of Sirius Red–positive areas in the annotated areas in Excel files.

Exposure to the Illuminant of a Whole Slide Scanner

To examine the effect of exposure by the illuminant of the WSS, we repeated scanning the specimens with WSS 30 times using the same method as mentioned in the paragraph about scanning the specimen images with WSS.

Following photography in JPEG format, the images were converted into grayscale by Adobe Photoshop CS6. They measured the grayscale values in specimen areas with the same method mentioned above in the paragraph on examining the effects of UV for heteropoly acid staining.

Tissue Analysis by Polarizing Microscope

The photographs of the Sirius Red–stained tissue section were taken at ×40 or ×100 magnification using an optical microscope (BX51; Olympus, Tokyo, Japan) attached with a digital camera (DP-22; Olympus) and image-capture software (cellSens Standard; Olympus). Following are the conditions: manual white balance; manual focus; sensitivity, ISO 200; exposure compensation, 1/3 (polarized condition) or 1 (non-polarized condition); resolution, 1920 × 1440. We considered a blank area of the glass slide for manual white balance as the background white balance area. For polarized observation, we inserted a DIC prism into the lens barrel and set the polarizer to the filter holder and rotated the polarizer clockwise until tissue sections were polarized and took images. The images were saved as a TIFF file (1920 × 1140 pixels). In ×100 magnification, we used immersion oil (IMMOIL-8CC; Olympus). We used the function of Adobe Photoshop CS6 for image overlay onto bright-field images. Non-polarized Sirius Red–positive area was selected by color pick-up function. Then, the picked up color was changed to dark blue (hue: −144, saturation: +41, and brightness: −22 for human samples; hue: −111, saturation: +51, and brightness: −38 for murine samples). New black and white layers were created for the remaining non-selected areas, thereby keeping the selected area function working. Thus, the remaining non-selected area color alone was abolished (i.e., grayscale). The new layer was created and the bright-field image was mounted onto the new layer. The opacity of the bright-field layer was changed to 40%. Then, all three layers were integrated.

Statistical Analysis

Statistical analysis was carried out using JMP Pro 15 software (SAS; Tokyo, Japan).

Tukey–Kramer’s honestly significant difference comparison of the mean value among multi-group and multiple nonparametric comparisons among multi-group were analyzed by Steel-Dwass.

P value was under 0.05, indicating a significant difference in each analysis.

Results

PTA Pretreatment Restrains the Nonspecific Nuclear Staining of PSR Staining and PMA Pretreatment Likewise

Previously, we reported that the NS observed in the PSR specimen was prevented by PMA + PSR staining. ²⁰ Then, we further analyzed the effect of pretreatment by PTA, categorized as the same heteropoly acids as PMA, in PSR staining for nonspecific NS. Representative images of PSR or PTA + PSR staining are shown in Fig. 1A. In Fig. 1B, we have summarized the data of 85 images or 90 images (5 images in a case with 17 cases or 18 cases) of the non-cancerous area of 17 cases of primary liver cancer or 1 of 18 cases of metastatic liver cancer. Distribution of Ishak score in the non-cancerous area of 17 cases of primary liver cancer was as follows: score 1: 1 case, score 2: 2 cases, score 3: 7 cases, score 4: 3 cases, score 5: 3 cases, score 6: 1 case. In PSR staining, NS was observed in part of the specimens—primary liver cancer, 12 images/85 images (14.1%); metastatic liver cancer, 5 images/90 images (5.6%), whereas in PTA + PSR staining, NS was never observed—primary liver cancer, 0 image/85 images (0%); metastatic liver cancer, 0 image/90 images (0%). In addition, we found that the pale red tone on the yellow color of the cytoplasm of the hepatocytes in PSR staining disappeared in the PTA + PSR condition (case 10 in Fig. 1A).

Figure 1.

Representative images and rate of emergence of the NS in PSR and PSR with PTA pretreatment. (A) The upper diagram is the representative case that does not have nonspecific NS (case 16 of the non-cancerous part of primary liver cancer). At the same time, the lower one is the case with nonspecific NS (case 10 of the non-cancerous part of the primary liver cancer) in PSR staining. Arrows indicate nonspecific NS. (B) The ratio of nonspecific NS observed cases in the non-cancerous part of primary and metastatic liver cancer in PSR and PTA-PSR staining. The upper part of each column indicates the number of cases with nonspecific NS (at least one image out of the five images in each case has nonspecific NS). In contrast, the lower part of each column indicates the number of images showing nonspecific NS out of a total of 85 images or 90 images in 17 cases or 18 cases. (original image: ×40, bar = 100 μm). Abbreviations: NS, nuclear staining; PSR, picrosirius red; PTA, phosphotungstic acid.

Thus, from the above results, it was suggested that pretreatment using PTA, one of the heteropoly acids, would restrain not only the nonspecific NS of Sirius Red but also the nonspecific staining of Sirius Red on the hepatocyte with PMA pretreatment.

Next, we examined Sirius Red–positive areas with the images used in Fig. 1 by image analysis software (Fig. 2). The percentage of Sirius Red–positive areas was analyzed with two-color range conditions for PSR staining. That is, we used two settings of color range: one setting never detected NS in any cases [Nuclear detection (Never)], and the other one detected maximum collagen fiber areas regardless of the NS [Collagen detection (Max)]. In the case of the PTA pretreatment condition, we used a color range setting that detects possible maximum collagen fiber areas [collagen detection (Max)] (Fig. 2A). The average of Sirius Red–positive areas in primary liver cancer was 0.017%, and the one of metastatic liver cancer was 0.025% in PSR staining of “Nuclear detection (Never)” condition. In comparing the “Nuclear detection (Never)” condition and “Collagen detection (Max)” condition in PSR staining, there were significant differences between the two groups in primary liver cancer cases and metastatic liver cancer cases: Nuclear detection (Never) vs collagen detection (Max), p<0.0001 (primary liver cancer cases) and Nuclear detection (Never) vs collagen detection (Max), p<0.0001 (metastatic liver cancer), whereas in PTA + PSR staining, the average Sirius Red–positive area in primary liver cancer was 0.714% and the one in metastatic liver cancer was 0.977%. There was a significant difference between PSR in “nuclear detection never” condition and PTA + PSR “collagen detection Max” condition; primary—PSR (Nuclear detection [Never]) vs PTA + PSR, p<0.0001; metastatic—PSR [Nuclear detection (Never)] vs PTA + PSR, p<0.0001 (Fig. 2B). In addition to the case of PSR staining, we compared the Collagen detection (Max) condition in PSR with the Collagen detection (Max) condition in PTA + PSR. The Collagen detection (Max) condition in PSR for primary liver cancer was 1.044% and the one for metastatic liver cancer was 1.563%. There was no significant difference in primary liver cancer cases or metastatic liver cancer cases between Collagen detection (Max) condition in PSR and Collagen detection (Max) condition in PTA + PSR: PSR vs PTA + PSR (primary liver cancer cases), p=0.5994 and NS + PSR vs PTA+PSR, p=0.3368 (metastatic liver cancer cases).

Figure 2.

Effect of stainability of the NS for detecting Sirius Red–positive areas in image analysis. (A) Representative analysis results for Sirius Red–positive areas in PSR and PTA-PSR were indicated (case 15). Arrows in the images are part of capturing the color of the NS as positive areas for image analysis. (a) Results under the detecting condition of Sirius Red–positive area without detecting nuclear staining in Sirius Red–alone staining, (b) results with the maximum Sirius Red–positive area detection regardless of the NS in Sirius Red–alone staining, and (c) results with the maximum Sirius Red–positive area detection in PTA-PSR staining. (B) Comparison of positive rate in the whole image of Sirius Red–positive areas for the non-cancerous part of the primary and metastatic liver cancer with setting conditions (A). In the box plot, × is the mean value, the line is the median, and the box’s up and down is the box’s quartile point (original image: ×40, bar = 50 μm). Abbreviations: NS, nuclear staining; PSR, picrosirius red; PTA, phosphotungstic acid.

These data suggested that the avoidance of nonspecific NS detection in PSR alone could not maximally detect collagen-positive areas given nuclear staining of avoidance detection. On the other hand, the positive area results obtained by Collagen detection (Max) condition in PSR-alone condition did not properly reflect the exact collagen-positive area due to detection of nonspecific NS. In contrast, PTA pretreatment could detect collagen-positive areas to a maximum extent without detecting nonspecific nuclear staining. Thus, PTA + PSR staining would be suitable for detecting collagen fibers in the whole area of the liver specimens.

PTA Treatment Does Not Lead to Blue Coloration Following WSS Light Exposure Although Slight Blue Coloration Is Observed Following UV Light Exposure

We previously reported that the color of specimens stained with PMA turned blue by exposure not only to UV but also to WSS light. ²⁰ Although PMA and PTA are categorized into heteropoly acids and are similar structures, ²⁵ we examined the effects of PTA on exposure to UV and WSS light. We used UV-cut film as the control to block UV light from tissue sections. The change of color tone in PMA- and PTA-stained specimens with or without UV-cut film by UV exposure is shown in Fig. 3A. The color image of the specimens was converted to a gray tone and the change in color tone was evaluated based on brightness to evaluate the depth of blue color on the specimens because of the blue coloration after UV or WSS light exposure. The value of gray tone in the case of PMA staining–alone specimens without UV-cut film compared with other conditions showed a statistically significant difference (i.e., turning color), whereas in the case of PTA staining alone, specimens appeared to slightly turn blue with the naked eye after exposure to UV. However, there was no significant difference among the values of gray of other groups except for PMA specimens exposed to UV by statistical analysis (data not shown).

Figure 3.

Investigation of color changes in human and mouse specimens under UV or whole slide scanner (WSS) light exposure. The upper figure shows the average gray values of grayscale-converted images of unstained, PMA-stained, and PTA-stained liver tissue specimens exposed to UV or WSS light. (A) The change in the color tone of sections stained only with PMA or PTA when exposed to UV light is shown as the average gray value. (B) The change in the color tone of sections stained only with PMA or PTA when exposed to WSS light is shown as the average gray value. The figure at the bottom shows the average gray values of grayscale-converted images of unstained, PMA-stained, and PTA-stained liver tissue specimens exposed to UV or WSS light for mouse specimens. (C) The change in the color tone of unstained, PMA-stained, and PTA-stained sections when exposed to UV light are shown as the average gray value. (D) The color tone changes in unstained, PMA-stained, and PTA-stained sections exposed to WSS light are shown as average gray values. In the box plots, × indicates the mean, the line in the box indicates the median, and the upper and lower sides of the box indicate the quartiles. *p<0.05. **p<0.0001. B: Before the UV or WSS light exposure; A: After the UV or WSS light exposure. Abbreviations: UV, ultraviolet; WSS, whole slide scanner; PMA, phosphomolybdic acid; PTA, phosphotungstic acid.

Next, we examined whether the color of specimens turns blue by exposure to WSS light (Fig. 3B). By 30 times exposure to WSS light, the gray tone value in PMA-stained specimen was significantly decreased. However, the one in PTA-stained specimen was not changed compared with pre-exposure specimens of any conditions as well as no stain specimen (PMA WSS light 30 times vs no staining pre-exposure: p<0.0001, PMA WSS light 30 times vs no staining WSS light 30 times: p<0.0001, PMA WSS light 30 times vs PMA pre-exposure: p<0.0001, PMA WSS light 30 times vs PTA pre-exposure: p<0.0001, PMA WSS light 30 times vs PTA WSS light 30 times: p<0.0001).

We further examined murine liver specimens to confirm whether the phenomenon observed in human samples would also occur in murine samples, that is, we conducted UV and WSS light exposure experiments on murine liver specimens. Exposure to UV light and WSS light (30 times) turned the color of PMA-treated specimens to blue. In contrast, PTA-treated specimens were not affected by exposure to UV light and WSS light (30 times) (Fig. 3C and D).

Thus, similar to PMA, PTA itself was possibly affected by UV exposure, and the color of specimens would change slightly. However, compared with PMA, PTA was not affected by WSS light exposure, and the specimen’s color was not changed at all. Thus, PTA treatment would be usable for the specimens, the digital data of which would be taken by WSS.

Evaluation of human liver fibrosis by Ishak score showed the best correlation with PTA + PSR.

We then analyzed the whole slide data to determine the percentage of Sirius Red–positive area in human non-tumor areas in primary liver cancer cases and then compared the evaluation with that using the Ishak score determined by the three pathologists (Figs. 4A–C). The correlation coefficients were as follows: PSR vs Ishak score: R = 0.54; PTA + PSR vs Ishak score: R = 0.64; and PMA + PSR vs Ishak score: R = 0.48. Then, we analyzed the effect of nonspecific NS on the percentage of Sirius Red–positive area in PSR staining. We compared the percentages of Sirius Red–positive area between the PSR and PTA + PSR conditions. The percentages of Sirius Red–positive area of three nonspecific NS-positive cases were plotted in the upper area of the regression line (Fig. 4D). We also compared the effect of color changes due to WSS exposure time on the detection percentage of the Sirius Red–positive area under PMA + PSR. We prepared two detection settings and compared their percentages of Sirius Red–positive area. The percentage of Sirius Red–positive area was clearly affected by WSS exposure time. For setting 1, the percentage of Sirius Red–positive area increased with multiple exposure, whereas for setting 2, the percentage of Sirius Red–positive area decreased with multiple exposure.

Figure 4.

Investigation of the correlation between the Ishak score and the percentage of Sirius Red–positive area under each staining condition, the effect on the evaluation of nonspecific NS in PSR staining, and the effect of the difference in the number of times of exposure to WSS light in PMA + PSR–stained same specimens for Sirius Red–positive region detection. (A) Correlation between the Sirius Red–positive rate of PSR staining and the Ishak score. (B) Correlation between the Sirius Red positive rate of PTA + PSR staining and the Ishak score. (C) Correlation between the Sirius Red–positive rate of PMA + PSR staining and the Ishak score. (D) Correlation between PSR staining and the Sirius Red–positive rate in PTA-pretreated PSR-stained specimens. Orange plots indicate cases in which PSR staining showed nonspecific NS. (E) Reversal phenomenon of analysis results of Sirius Red–positive regions in PMA-pretreated PSR-stained specimens due to differences in the number of WSS light exposure of the same specimen. Abbreviations: NS, nuclear staining; PSR, picrosirius red; WSS, whole slide scanner; PMA: phosphomolybdic acid; PTA: phosphotungstic acid.

These results suggest that the percentage of Sirius Red–positive area is affected by nonspecific NS in PSR. Thus, the data are biased by nonspecific NS in PSR. In addition, our results also suggested that the percentage of Sirius Red–positive area in PMA + PSR condition is affected by WSS light exposure time. Thus, the percentage of Sirius Red–positive area is affected by WSS light exposure time in PMA + PSR condition.

PTA + PSR Affect the Staining of Collagen Fibers

We compared the average percentage of Sirius Red–positive areas between WSS-scanned data on human non-tumor areas of primary liver cancer cases and murine liver tissues. In human specimens, the average percentage of Sirius Red–positive area for both PTA + PSR (PSR vs PTA + PSR: 10.220% vs 6.929%; p=0.0165) and PMA + PSR (PSR vs PMA + PSR: 10.220% vs 4.372%; p<0.0001) was significantly lower than that for PSR (Fig. 5A).

Figure 5.

Comparative study of the average percentage of Sirius Red–positive area under various staining conditions in human and mouse tissues. (A) Average percentage of Sirius Red–positive area under various staining conditions in non-cancerous liver tissues of human primary liver cancer cases. (B) Average percentage of Sirius Red–positive area in MDR2 mouse liver tissue. In box plots, × indicates the mean, the line in the box indicates the median, and the upper and lower sides of the box indicate the quartiles. *p<0.05. **p< 0.0001. Abbreviations: PMA: phosphomolybdic acid; PSR, picrosirius red; PTA: phosphotungstic acid.

In murine liver tissue, we compared whole specimen liver WS data for the percentage of Sirius Red–positive area in PSR, PTA + PSR, and PMA + PSR. Compared with PSR, the average percentage of Sirius Red–positive area for PTA + PSR (PSR vs PTA + PSR: 0.765 vs 0.398%; p=0.0007) and PMA + PSR (PSR vs PMA + PSR: 0.765% vs 0.570%; p=0.0171) also significantly decreased (Fig. 5B). To determine the reasons for the decrease in percentage of Sirius Red–positive area for PTA + PSR condition, we analyzed Sirius Red–stained specimens with polarized microscopy. We examined specimens used in Figs. 1 and 2 in this analysis. Representative results of the PSR-alone condition and PTA-pretreated condition are shown in Figs. 6 and 7 for human and murine cases, respectively. Non-polarized Sirius Red–positive substances, localized surroundings of straight-lined polarized collagen or reticular fibers, were observed in PSR condition alone (Figs. 6A and 7A). The amount of non-polarized Sirius Red–positive substances was decreased for the PTA + PSR condition (Figs. 6B and 7B), which allowed for the easy detection of polarized collagen/reticular fiber, indicating that PTA pretreatment did not reduce specific staining for collagen or reticular fibers in both human and murine cases. Finally, we tried to overlay the bright-field image with pseudo-colored non-polarized Sirius Red–positive substances in polarized images to see how non-polarized Sirius Red–positive substances appear in bright-field images (Figs. 6E, F and 7E, F). We found that non-polarized Sirius Red–positive substances were abundant in bright-field images where Sirius Red is heavily stained in PSR condition.

Figure 6.

Comparison of PSR and PTA-pretreated PSR-stained human specimens between bright-field images. The images of human primary liver cancer (case 16). The upper diagram (A and B) shows the bright-field condition; the middle diagram (C and D) shows the polarized condition; the lower diagram (E and F) is an overlay image of opacity-decreased bright-field image (A and B) onto the polarized image (C and D) in which pseudo-color was given (dark blue) to Sirius Red–stained non-polarized area (original image: ×40, bar = 100 μm). Abbreviations: PSR, picrosirius red; PTA: phosphotungstic acid.

Figure 7.

Comparison of PSR and PTA-pretreated PSR-stained murine specimens between bright-field images. The images of murine liver tissue. The upper diagram (A and B) shows the bright-field condition; the middle diagram (C and D) shows the polarized condition; the lower diagram (E and F) is an overlay image of opacity-decreased bright-field image (A and B) onto the polarized image (C and D) in which pseudo-color was given (dark blue) to Sirius Red–stained non-polarized area (original image: ×100, bar = 20 μm). Abbreviations: PSR, picrosirius red; PTA: phosphotungstic acid.

These data suggested that non-polarized Sirius Red–positive substances surrounding collagen or reticular fibers decreased by PTA pretreatment, thereby preserving the fiber stainability.

Discussion

First, we wanted to emphasize that we can analyze liver fibrosis by highly magnified WSS scanning of WS images of all areas in mouse and some areas in human cases. For this highly magnified WS image analysis, nonspecific nuclear staining in PSR-alone condition is one of the most critical barriers to analyzing Sirius Red–positive area. Until now, in pathological semi-quantification of liver fibrosis, Vincenza Calvaruso et al. ¹⁴ and Abe et al. ¹⁷ analyzed the CPA of loupe image for the whole slide area of the liver section, and they did not examine highly magnified images. Although Jimenez et al. ²⁶ analyzed liver fibrosis with highly magnified images, they did not use WS but did represent five magnified fields per case. The former was neglecting the small Sirius Red–positive area in the hepatic lobule, whereas the latter was selecting a small area; the analysis results would change due to the selected area of analysis. Analysis of WSS-scanned highly magnified WS data used in this study not only fulfilled that the targeted area was an entire area of the specimen but also fulfilled that the analyzed image was of high magnification. Indeed, Stuart Astbury et al. ⁹ performed a whole slide analysis using the highly magnified image of the liver section. However, Julia Schipke et al. ²⁷ reported a problem of Sirius Red staining with nonspecific NS during the detection of the collagen fiber due to the same tone of nonspecific NS with collagen fibers. Our method, PTA pretreatment, suppresses nonspecific NS and overcomes this problem. Thus, we consider this method revolutionary because we can analyze WS data at high magnifications regardless of the nonspecific NS.

Next, we discuss the difference between PMA and PTA as heteropoly acids. Usually, both are used as mordants before staining with orange G or fast green FCF when we pathologically perform specific staining of collagen fiber, such as Mallory or Masson’s trichrome staining. ²¹ Heteropoly acid is a complex of proton acids combined with the anion of polyoxometalate, whose basic structure constitutes octahedrons of metal and oxygen. Above all, it is known that the Keggin heteropoly anion has the structure of XM₁₂O₄₀^y− (X is the central atom like Si⁴⁺, P⁵⁺, etc.; y is a negatively charged cluster; M is the metallic ion like Mo⁶⁺ and W⁶⁺). ²³ It is said that these heteropoly acids show very strong acidity in aqueous solution and combine with protein and amino acid, but an affinity for the nucleus is very low.

In comparison, it is said that PTA and PMA firmly combine with the collagen fiber, but are less combined in the cytoplasm. ²² Therefore, the above facts indicated that heteropoly acids would not directly combine in the nucleus to prevent the stainability of Sirius Red dye. In contrast, it is known that PTA and PMA treatment for specimens affect the stainability of anionic dye. For example, PTA and PMA suppress the stainability of anionic dye constituted of small molecules. ²⁸ The molecular weight is small in terms of picric acid (MW: 299). Thus, it is possible that the decreased yellow color of hepatic cells by picric acid would be induced by PTA pretreatment.

In contrast, it is also known that PMA and PTA suppress stainability of large-molecule dye such as aniline blue (MW: 800) to cytoplasm and non-collagenous fiber components. Because Sirius Red is an anionic dye and a large molecule (MW: 1372), PTA and PMA pretreatment probably influenced the stainability of Sirius Red to non-collagenous components, including the nucleus. Actually, in our polarized microscopic observation, stainability of non-polarized Sirius Red–positive component (which was not collagen or reticular fiber) was prevented by PTA pretreatment. However, the specific staining to collagen or reticular fiber was not affected.

Third, we would like to discuss what is stained as a non-polarized Sirius Red–positive component clinging to fiber. Collagen fiber is rich in basic amino acids and responds to acidic dye strongly. ⁴ Sirius Red is a strongly anionic long molecule that responds to collagen fiber via sulfonic acid groups and induces the polarization of collagen fibers due to many dyed molecules arranged parallel at the long-axis direction of collagen fibers. ⁸ Besides, there are hyaluronic acid, chondroitin sulfate, and heparan sulfate surrounding collagen fibers, and all of them exist in the liver, and heparan sulfate is the richest.^29,30 As the preliminary experiment, we examined how stainability of Sirius Red decreases by hyaluronidase, chondroitinase, or heparanase treatment. However, we found no difference between enzyme-treated conditions and enzyme non-treated conditions (data not shown). Thus, we could not conclude that these components would be candidates of nonspecifically stained non-polarized components clinging to fiber.

Finally, we discuss the sensitivity of heteropoly acid to light. PMA, as we previously reported, turns blue to form molybdenum blue by exposure to UV or blue light. ²⁰ Also, it is known that PTA slightly turns blue by UV light. ³¹ These changes can be considered as heteropoly acids, such as PMA and PTA, becoming heteropoly blue by UV light. ³² In histological examination, it is said that heteropoly blue is formed when the tissue stained with heteropoly acids, such as PMA and PTA, is treated by titanium(III)sulfate and stannous chloride. ³³ However, to the best of our knowledge, no paper shows tungsten blue is formed by UV light when the tissue is stained with PTA, as we have done in this study. Thus, our results would be noteworthy to be reported. Although it is known that PTA, one of the heteropoly blues, changes to tungsten blue by visible light in an aqueous solution in the presence of titanium oxide. ³⁴ However, in this study, we proved that PTA treatment to liver specimen did not produce tungsten blue by 30 times exposure to WSS light in which UV component was never present as we had previously reported. ²⁰ It is possible that there were no reducing agents such as titanium oxide in the non-aqueous embedded liver tissue to change PTA to tungsten blue. In this study, we developed PTA-pretreated PSR staining method that could suppress nonspecific NS and nonspecific non-polarized substance staining without disturbing specific collagen fiber staining. In addition, our method did not produce tungsten blue using a WSS, which enables us to analyze WSS-scanned highly magnified whole slide image data by CAIA. Thus, our method increases the accuracy of quantification and evaluation of liver fibrosis in histological examination.