Elsevier

Cardiovascular Pathology

Volume 13, Issue 4, July–August 2004, Pages 213-220
Cardiovascular Pathology

Original Article
A modification of the staining technique of reticular fibres for image analysis of the cardiac collagen network

https://doi.org/10.1016/S1054-8807(03)00153-4Get rights and content

Abstract

Introduction

Silver stain of reticular fibres demonstrates the fine structure of the cardiac collagen network. However, nuclei are also stained with current techniques, a drawback that makes computer image analysis difficult. To solve this problem our study was designed to modify Gomori's method. Reactive concentrations and action times represent the core of that modification. Only stromal tissue is stained. The technique was tested for repeatability and reproducibility to assess the precision of the measurement procedure in the assessment of myocardial collagen in a consecutive series of myocardial samples from patients with and without heart muscle disease. In addition, we checked the reliability of the method by comparing our results with the point-counting method (PCM).

Methods

The right ventricular myocardium was fixed in 10% buffered formaldehyde and routinely processed. Paraffin sections (4 μm) were stained with several modifications of Gomori's method. Variable concentrations of potassium permanganate, silver solutions and different oxidation times were tried. A field from each sample was digitized. Additionally, the technique was tested for repeatability and reproducibility.

Results

We obtained absence of background and nuclear staining together with a highly contrasted image of the collagen network with 3 min oxidation with 2% potassium permanganate and a silver concentration of 1% during 5 min. In this way, it was very easy to perform acquisition, thresholding and area measurement without any further manual processing of the image.

Conclusions

This technique appears very helpful for the quantitative study of the cardiac collagen network by means of computerized image analysis systems.

Introduction

Although the prevailing paradigm is that heart disease is largely explained in terms of muscle cells, one should not lose sight of the crucial importance of the extracellular matrix in the structure and function of the myocardium [1], [2], [3].

Light microscopy of the cardiac collagen network was studied by silver impregnation techniques [1], [4], [5], trichrome stains [6], picrosirius red technique [7], [8] and immunohistochemistry [9], [10].

Silver impregnation techniques are by no means specific for reticular fibres [11], [12]. They yield positive results reacting with material associated with reticular fibres (apparently proteoglycans) rather than with the fibres themselves. However, silver stains for reticulum demonstrate with high accuracy the fine structure of the collagen components of the myocardium (endomysium, perimysium and epimysium) while being more sensitive than usual trichrome stains [4], [5].

The usual technique for the investigation of reticular fibres in the myocardium is a modification of the silver-staining method of Del Rio-Hortega developed by Robinson et al. [5]. The latter technique, in spite of being excellent for myocardial stroma, requires frozen sections of 80–120 μm (too thick for a correct image analysis). Additionally, it does not permit a prospective study of tissue embedding in paraffin.

There are several silver techniques for paraffin sections [13], [14]. A serious drawback of the usual techniques for silver staining is that generally the nuclei and background are also stained (Fig. 1). This makes computer image analysis difficult and by no means capable of automatic thresholding. Manual exclusion of background and nuclear area is required. To solve this problem the present study was designed to modify the oxidation time and the concentration of silver solutions in order to eliminate background and nucleus staining. Image analysis is thus permitted.

Our technique was tested for repeatability and reproducibility to assess the precision of the measurement procedure. If it is not repeatable (large variability across trials) or reproducible (large variability across observers) relative to the variability between stained slides, the measurements will not be precise enough for routine histopathological applications. We also compared the results obtained with computer-based image analysis method (IAM) with the classic and standardized quantitative point-counting method (PCM) for determining the level of consistency or agreement between both methods.

The importance of method standardization may seem obvious, but must not be underestimated. In fact, Schulte [15] felt the need of dedicating relevant and wise reflections on this problem. On one hand, standardization of dyes and staining procedures may help to reduce inconsistent stain quality. This may help to avoid one of the most disturbing practical problems for the user, namely, poor staining due to poor stains. On the other hand, recent developments in computer technology permit the use of high-resolution computer image analysis with cytological and histological material. These techniques require highly reproducible staining patterns.

The aim of the present paper is the standardization of Gomori's stain technique for quantification of collagen in the heart extracellular matrix of normal and pathologic specimens with computerized image analysis.

Section snippets

Specimens

Tissue blocks from the right ventricular outflow tract were taken from five normal hearts, according to standard autopsy procedures of sampling the heart [16], [17]. These hearts were obtained from the Homograft Bank of the Hospital de Pediatrı́a Prof. Dr. Juan P. Garrahan. Donors were four males and a female; ages and time interval between death and autopsy are shown in Table 1. Each block was examined showed no macroscopical or major light microscopical abnormalities.

To test if the technique

Staining

The results of the different modifications are shown in Table 2. Each variant was evaluated histologically for the presence of background, myocyte nuclear or cytoplasmic staining and the quality of the staining of the collagen framework.

With low concentrations and short times of oxidation, the background was increased and the staining of the coarse collagen fibres was more intense than the staining of both thin fibres and nuclei. With long times of oxidation, background and nuclear staining was

Discussion

Picrosirius red, trichrome stains and immunohistochemical techniques were used for computerized image analysis of the cardiac collagen network [6], [7], [8], [9], [10]. As far as we know, silver stains, despite having been extensively used for characterization of the cardiac stroma, were not adopted for quantification through image analysis.

One of the most important goals of the digital image analysis was to develop a specific technique for analyzing the tissue components [18]. This was aimed

Summary

Silver staining of reticular fibres accurately demonstrates the fine structure of myocardial collagen. A drawback of silver stains with the usual techniques consists of the nuclei being also stained, making automated image analysis difficult. This paper reports a modification that avoids that drawback and allows quantification of heart collagen in normal and pathologic heart muscle with computerized image analysis.

Acknowledgements

This study was supported by the Morón University Foundation. The authors express their gratitude to Dr. Jorge M. Affanni for their editorial assistance in the preparation of this manuscript.

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