Improving light microscopic detection of collagen by trichrome stain modification

Improving light microscopic detection of collagen

by trichrome stain modification

S.S. Al-Mahmood

Department of Pathology and Poultry diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Email: saevan981@yhaoo.com

Abstract

In present study we aimed to introduce modifications in classical protocol applied to stain collagen fibers by Masson's trichrome stain, in order to decrease time of different steps and concentration of chemicals in this traditional protocol. The experiment design contains a series of successive amendment steps based on previously successful steps, in which every step where be modified to get the best result, then the next step of staining protocol will be modified in the classical Masson's trichrome staining protocol. A tissue sections from liver, heart and intestines of sheep diagnosed with chronic fasciolosis were selected to be used in current study. The result showed that the new modified protocol gives the same staining efficiency to collagen fiber when Harris hematoxylin used as a nuclear strainer or even excluded this stain from staining protocol. In conclusion this new modified staining protocol give a perfect staining reaction to collagen fibers in pathological samples which is similar to that obtained by traditional Masson's trichrome stain, also this new modified procedure is less time consuming and less toxic effect on human and environment than other trichrome stains, and can be easily conducted either by the technician or the pathologists.

Keywords: Collagen, Modified, Trichrome, Improving, Histopathology

DI: 10.33899/ijvs.2020.126176.1256, ©2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

تحسین الکشف بالمجهر الضوئی عن الکولاجین من خلال تحویر ملون ثلاثی اللون

سیڤان سعد المحمود

فرع الأمراض وأمراض الدواجن، کلیة الطب البیطری، جامعة الموصل، الموصل، العراق

الخلاصة

هدفنا فی الدراسة الحالیة إحداث تحویرات فی التقنیة الکلاسیکیة المتبعة لتلوین الیاف الکولاجین باستخدام ملون ماسون ثلاثی اللون، وذلک من اجل تقلیل الزمن والترکیز للخطوات المتتابعة والمواد الکیمیائیة فی التقنیة التقلیدیة. احتوى تصمیم التجربة على إحداث تغیرات ناجحة متتابعة فی مرحلة معینة معتمدة على التحویرات الناجحة فی الخطوات السابقة للملون التقلیدی لماسون ثلاثی اللون. مقاطه نسجیة لکل من الکبد، والقلب، والأمعاء لأغنام مصابة بداء المورقات المزمن استخدمت فی هذه الدراسة. أظهرت النتائج لتقنیة الملون المحور الجدید أنها أعطت نتائج مشابهة لکفاءة تلوین الیاف الکولاجین عند استخدام ملون هاریس الهیماتوکسیلین کملون للنواة أو حتى بدون استخدام الهیماتوکسیلین فی خطوات التلوین. یمکن الاستنتاج أن التقنیة الجدیدة المحورة أعطت تفاعل تلوینی جید لألیاف الکولاجین فی العینات المرضیة مکافئة لتلک النتائج التی تم الحصول علیها عند التلوین بملون الماسون ثلاثی اللون التقلیدی، فضلاً عن أن هذه التقنیة المحورة الجدیدة للتلوین کانت اقل استهلاکاً للوقت واقل سمیة للإنسان والبیئة من الملون ثلاثی اللون لماسون، ویمکن بسهولة إجرائها من قبل الفنی الإمراضی أو الإمراضی نفسه.

Introduction

Histology can be defined as the science that study the normal composition of tissues, while histological stains consider the key stone that used till now to explain different component of tissues (1). Histology and stains were invented and developed after a huge leap done by Antonie van Leeuwenhoek by invention the simple microscopy (2). Hematoxylin and Eosin stain (HE) and its modified protocols considered the main stain that applied routinely to visualize the components of tissues, in contrast, a special stain lead to expression a specific tissue component either in intracellular or extracellular location that could not clearly distinguished during HE routine staining (3). One of these components that need distinctive differentiation from surrounding cellular element is collagen fibers, these fibers are produced by mature fibrocytes and fibroblast in response to wide range of inflammatory cytokines produced by normal physiological and pathological process (4). The most common special stain used to facilitate the observation and identification of collagen fibers is trichrome stain, specifically Masson's trichrome stain, this stain and its modified protocols are widely used all over the global laboratories (4). Trichrome stain is invented by French scientist Claude L. Masson at 1910s, who used this stain to differentiate between collagen, muscle, and other fibers (3). Briefly, the original Masson's trichrome stain composed from four main stains, (i) Wiegert hematoxylin used to stain nucleus, (ii) Acid stain which target to stain muscles and red blood cells, (iii) Phosphatic acids that used to blech previous stains from collagen fibers and work also as mordent to anionic dyes that stain collagen in next step, and (iv) Collagen fiber stain (either Aniline Blue or Light Green SPF) that can be used to accomplish the goals of Masson's trichrome technique (3). Like all other special stains, this technique has undergone many modifications which aim to improving the accuracy and efficiency of collagen fibers staining (5). Since this special stain contain many toxic even carcinogenic chemical materials in addition time consuming protocol, this study designed to eliminate these hazardous chemicals and decrease their concentrations, in addition, to improve the detection of collagen fibers using a new modified technique of Masson's trichrome stain.

Materials and methods

Tissue sampling and processing

A tissue samples were collected from liver, heart and intestines of sheep diagnosed with chronic fasciolosis. The tissue samples fixed in 10% neutral buffered formalin for at least 72 hours, then a represented sample were collected from area showed gross appearance of fibrosis, sliced at thickness less than 7 mm, later these samples labeled with specific code, dehydrated with ascending concentration of ethyl alcohol, cleared in xylene, infiltrated and embedded with hot paraffine wax at 56ºC, later tissue blocks were sectioned at 6 µm thickness, lifted at glass slides, attached to slide at 56±2ºC for one hour, left to cool for another one hour, then cleared with xylene, rehydrated with descending ethyl alcohol to distilled water (6).

Routine and special stain

The histopathological glass slides from previous steps were stain by routine Harris hematoxylin and alcoholic red eosin (3) in order to confirm classical lesions of chronic fasciolosis and fibrosis, also the classical Masson's trichrome stain was used to stain slide which described by Layton and Bancroft (4) with Aniline Blue, slides with these stains considered as a control slides.

Experiment designs

The experiment design contains a series of successive amendment steps based on previously successful steps, in which every step where be modified to get the best result, then the next step of staining protocol will be modified in compare with the classical Masson's trichrome staining protocol.

Step one (mordent with Bouin's Solution) Bouin's solution used as a mordent for tissue samples fixed in formalin fixative (4), in this step the length of fixation with Bouin's solution will be decreased at a regular interval of 5 minutes in order to reach the least time for mordent which gives similar results to the control group.

Step two (staining the nucleus) Wiegert's hematoxylin used in Masson's trichrome stain in order to stain the nucleus (4), this stain will be modified in two manner (i) slides stained with Harris' hematoxylin instead of Wiegert's hematoxylin, and (ii) slides where not stained with hematoxylin stain.

Step three (staining with acidic stain) an equal mixed solution of 1% acid fuchsine and 1% biebrich scarlet where used as acidic dye in Masson's trichrome stain, in which the biebrich scarlet will stain the red blood cells, while acid fuchsine will stain muscle fibers (4), in this step the biebrich scarlet will be excluded from the staining steps, and the acid fuchsine will be decreased in concentration and staining time.

Step four (phosphate acids) both phosphomolybdic and phosphotungastic acid are the most commonly used in Masson's trichrome stain, they bind to collagen fibers and act as a site of conjunction with collagen stain that applied later (4), this step will be modified by decreasing the concentration and exposure time to phosphatic acids.

Step five (collagen stain) aniline blue is used to stain collagen fibers that conjugated with phosphatic acids, in which the anionic stain will attach to phosphatic acids rapidly due to negative charge induced by phosphatic acids (4), this step will be modified by decreasing the concentration and staining time to aniline blue.

Evaluation of collagen fibers staining efficiency

Tissue slides that stained in current study were examined by light microscope at 40x, using ToupView software version 1.0 to measure the intensity of collagen fibers staining (blue color). In brief, five similar fields in all slides from the same organ were subject to image color evaluating process, in which bright blue color were considered the positive result, then the image process calculated the mean of bright blue color in each field (7), later, the mean were statistical analyzed with aid of SPSS software version 19.0 at P

Results

Modified steps of trichrome stain (plates 1, 2, 3, and 4)

Mordent with Bouin's Solution

The result of this step showed that, decreasing in the time of fixation in Bouin's solution have a similar result to control slides when Bouin's solution preheated to 55 ºC and slides fixed for 15 minutes at 60 ºC, else that slides where fixed in Bouin's solution less than 15 minutes showed losing of brightness of collagen.

Staining the nucleus

The result of this step showed that, staining of slide with Weigert's hematoxylin or Harris hematoxylin or even without staining by hematoxylin stains did not affect the staining properties of collagen stain.

Instead, slides that not stained with hematoxylin stain are better in illustration of collagen fibers stained with Aniline Blue.

Staining with Acidic stain

The result of this step showed that, excluding of Biebrich Scarlet stain did not affect the staining properties of collagen or general architecture of stain, and reduction of Acid Fuchsine concentration to 0.1% for one minute will give the same result of muscle fibers and red blood cells as same as in control group.

Phosphatic Acids

The result of this step showed that, decrease the concentration of aqueous solution of Phosphomolybdic and Phosphotungastic acid to 0.5% for 5 minutes will give the same result of staining properties to collagen fibers that have been recorded in slides of control group.

Collagen Stain

The result of this step showed that, the concentration of 0.2% of aqueous solution of Aniline Blue give perfect staining properties to collagen fibers when used for one minute.

Evaluation of collagen fibers staining efficiency

The result of current study related to efficiency of collagen fiber staining showed that there are no significant differences between the slides stained with Masson's trichrome stain (control group) and slides stained with modified Masson's trichrome stain (experimental groups), these result indicate that the new modified staining protocol were have the same staining efficiency that obtained by classical protocol for Masson's trichrome stain (Table 1).

Table 1: Evaluation of collagen staining efficiency in control and experiment protocol

Letters different in column have a significant difference at P<0.05.

Saevan's modified - Masson’s trichrome stain protocol

Solutions

Bouin's solution prepared by mixing 75 ml of formaldehyde 37%, 25 ml of saturated aqueous solution of Picric acid, and 1 ml of acetic acid (Glacial).

Aniline Blue stain prepared by mixing aniline Blue, 0.2 gram in 100 ml of distilled water, the added 1 ml of acetic acid (Glacial).

Acid fuchsine satin prepared by mixing acid fuchsine, 0.1 gram, in 100 ml of distilled water.

Phosphomolybdic - Phosphotungastic acid solution prepared by mixing 0.5 grams of phosphomolybdic acid, with 0.5 grams of phosphotungastic acid, in 100 ml of distilled water, the added a 1 ml of acetic acid (Glacial).

Aqueous acetic acid solution prepared by adding 3 ml of acetic acid (Glacial), into 97 ml of distilled water.

Plate 1: Showed histopathological comparison between the whole modified protocol of trichrome stain and traditional Masson's trichrome stain in liver, heart and intestines of sheep diagnosed with chronic fasciolosis.

Plate 2: Showed histopathological comparison between the whole modified protocol of trichrome stain and traditional Masson's trichrome stain in liver of sheep diagnosed with chronic fasciolosis, in which the comparison conducted in (i) portal area, (ii) central vein, and (iii) collagen fibers.

Plate 3: Showed histopathological comparison between the whole modified protocol of trichrome stain and traditional Masson's trichrome stain in intestine of sheep diagnosed with chronic fasciolosis, in which the comparison conducted in (i) intestinal villi, (ii) muscular layer, and (iii) serous layer.

Plate 4: Showed histopathological comparison between the whole modified protocol of trichrome stain and traditional Masson's trichrome stain in heart of sheep diagnosed with chronic fasciolosis, in which the comparison conducted in (i) endocardium, (ii) myocardium, and (iii) epicardium.