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Utility of orcein stain and comparison with Masson trichrome stain in chronic intestinal pseudo-obstruction


 Department of Pathology and Lab Medicine, AIIMS, Bhubaneswar, Odisha, India

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Date of Submission31-May-2021
Date of Decision23-Nov-2021
Date of Acceptance15-Jul-2021
Date of Web Publication26-May-2022
 

   Abstract 

Chronic intestinal pseudo-obstruction (CIPO) is caused by a plethora of primary and secondary causes, dominantly involving the neuromuscular tissue, interstitial cells of Cajal, or the connective tissue framework. The lack of the connective tissue framework, known as desmosis, is evaluated by Masson's trichrome (MT) or picrosirius red stains, both of which are recommended in the London classification. We evaluated the orcein stain in detecting desmosis in comparison to the MT stain. We performed both orcein and MT stains in six previously published cases of complete or partial desmosis along with six age-matched controls. Our results showed comparable results of the orcein stain as compared to the MT stain. Additional advantages of lower cost and a clearer background in orcein stain were noteworthy, whereas MT stain can be used for the detection of additional pathology. We believe that orcein stain can be used as a cheap alternative in resource-limited settings.


How to cite this URL:
Ayyanar P, Mitra S. Utility of orcein stain and comparison with Masson trichrome stain in chronic intestinal pseudo-obstruction. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Jan 29]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=345882





   Introduction Top


Chronic intestinal pseudo-obstruction (CIPO) is caused by the functional lack of gastrointestinal peristalsis in the absence of any organic pathology. The causes are variable and dominantly attributed to the diseases affecting the smooth muscles, nerves and ganglia, interstitial cells of Cajal, and/or the connective tissue meshwork. The connective tissue meshwork is located within the circular and longitudinal muscle of the muscularis propria (intramuscular extension) and the plexus layer (at the level of the junction between circular and longitudinal layer and around the myenteric plexus) as a tendinous sling.[1] This sling is composed of type III collagen, which contracts and relaxes alternately with the function of the circular and longitudinal muscle layer. It anchors the smooth muscle cells and provides tensile strength. The total or focal lack of connective tissue meshwork is known as “desmosis,” which has been exclusively described in the colon. The recommended special stains for the detection of desmosis are trichrome, Van Gieson, or picrosirius red.[2] Most of the reported cases have mentioned the utility of these stains and immunohistochemistry for collagen subtypes. However, the literature is largely silent about the utility of orcein stain, a cheap vegetable stain that can be used to see the elastic fibers. Hence, we wanted to evaluate the utility of orcein stain in detecting the connective tissue meshwork in the normal colon as well as in desmosis.


   Materials and Methods Top


Orcein stain was performed on six cases of desmosis (four cases of complete desmosis and two cases of partial desmosis) associated with adult gastrointestinal inflammatory neuropathy. All these cases were previously reported by us.[3] It was also performed on the age-matched control sections of the colon obtained from the autopsy cases (n = 6) with good tissue preservation and lacking any gastrointestinal pathology. We compared the results of orcein stain with the results of Masson's trichrome (MT) stain.

Standard procedures were followed for both orcein and MT stains. Commercially available orcein stain (RM277, HiMedia Laboratories, Mumbai, India) was used. Briefly, the procedure of orcein stain required the oxidation in acidified potassium permanganate solution for 5 to 10 min following bringing the section to water. The oxidation step was followed by rinsing in tap water and subsequently distilled water. Thereafter, bleaching was performed in 2% oxalic acid for a minute and several changes of distilled water rinse were performed. The slides were then immersed in orcein stain solution (pH 1–2) for 4 h at room temperature, rinsed in 70% alcohol, and dehydrated and mounted for interpretation.


   Results Top


Orcein stain highlighted the tendinous scaffolds between the smooth muscle bundles of the circular and longitudinal layers and in the plexus layer in the control sections. All control sections showed the presence of tendinous sling as well-delineated, delicate, brownish-black elastic tissue fibers in a pale creamy background. Normal tendinous-sling meshwork has three components, namely a) longitudinal fibers that run parallel to each other within the inner circular layer as well as within the outer longitudinal muscles, b) transversely oriented fibrous condensation around the myenteric plexuses (peri-myenteric plexus fibers), and c) transversely oriented fibers connecting the myenteric plexuses (inter-myenteric fibers) lying in between the outer longitudinal and inner circular layers. The longitudinal sling within the outer longitudinal layer lies at a 45° angle with the transverse inter-myenteric fibers [Figure 1]. The density, nature (linear and few wavy fibers), thickness, and orientation (parallel, few haphazard arrangements) of these fibers in the control sections were comparable with MT stain. The visualization of elastic fibers was easier in orcein stain due to a clear background. The cases of desmosis showed comparable results with MT stain. [Figure 1] compares the orcein stain (1a, 1b) and MT stain (1c, 1d) in a normal colon, whereas [Figure 2] compares the above-mentioned stains in a case of partial desmosis. [Figure 3] compares both the stains in a case of complete desmosis.
Figure 1: Normal components of the tendinous-sling meshwork in the colon: longitudinal fibers (black arrows) running parallel to each other within the inner circular and the outer longitudinal muscle layers, peri-myenteric plexus transverse fibers (thick white arrows), and transversely oriented inter-myenteric fibers (dotted black arrows). Orcein stain (1a and 1b), Masson's trichrome stain (1c and 1d).

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Figure 2: Tendinous-sling meshwork in partial desmosis: the variable presence of only part of the tendinous sling. Either of the longitudinal fiber (black arrows), peri-myenteric plexus fiber (thick white arrows), or transverse inter-myenteric fiber (dotted black arrows) is absent. Orcein stain (2a and 2b), Masson's trichrome stain (2c and 2d).

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Figure 3: Tendinous-sling meshwork in complete desmosis: a complete absence of all parts of the tendinous sling. Orcein stain (3a and 3b), Masson's trichrome stain (3c and 3d).

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The major advantage of orcein stain in terms of visualization of the tendinous sling was darkly stained fibers in a clear background than the trichrome stain where bluish-green fibers were seen in a red background. Besides, orcein stain is cheaper (approximately INR 35.00/slide) than MT stain (approximately INR 45.00/slide). Orcein stain highlights the elastic laminae of the vessels aiding the assessment of the vasculopathic lesions. Nevertheless, MT stain can be used for the assessment of various myopathic and vasculopathic lesions. Besides, fibrosis can also be assessed in the trichrome stain.


   Discussion and Conclusion Top


The major histopathological counterparts of CIPO are neuropathy, myopathy, mesenchymopathy, and desmosis. The evaluation of desmosis requires careful estimation of the tendinous sling in trichrome stain to search for its absence. Both MT and picrosirius red are the standard stains for this purpose.[1],[2],[3] However, orcein, an elastic stain, can also be utilized for the same. The additional advantage of orcein as compared to MT is its clearer background and lower cost, whereas MT is invaluable for the simultaneous estimation of other morphological features.

The London classification of gastrointestinal neuromuscular pathology, an international working group (IWG 2009), recommends trichrome or picrosirius stain for the diagnosis of desmosis.[2] Mallick et al.[1] have mentioned that sirius red stain is better to demonstrate desmosis when compared to trichrome stain. Orcein stain is used to demonstrate elastic fibers in the connective tissue, copper-binding protein, hepatitis B surface antigen, and sulfated mucin.[4] Orcein stain highlights the elastic fibers in the connective tissue meshwork and can be helpful in the assessment of desmosis. This usage of orcein stain is not discussed in English literature to the best of our knowledge. Our observation may help the histopathologists in resource-limited settings.

Acknowledgments

The authors thank Mr. Samiur Raheman, technician-in-charge in the Department of Pathology and Laboratory Medicine, AIIMS, Bhubaneswar, for his assistance.

Contributorship statement

PA collected and analyzed the data and prepared the manuscript. SM conceptualized the idea, diagnosed the cases, and reviewed the manuscript.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Ethical Committee (T/IM-MF/Patho/19/68).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Mallick S, Prasenjit D, Prateek K, Shasanka PS, Virender S, Rajni Y, et al. Chronic intestinal pseudo-obstruction: Systematic histopathological approach can clinch vital clues. Virchows Arch 2014;464:529-37.  Back to cited text no. 1
    
2.
Knowles CH, De Giorgio R, Kapur RP, Bruder E, Farrugia G, Geboes K, et al. The London Classification of gastrointestinal neuromuscular pathology: Report on behalf of the Gastro 2009 International Working Group. Gut 2010;59:882-7.  Back to cited text no. 2
    
3.
Mitra S, Ayyanar P, Purkait S, Mishra SS, Mishra TS. Clinical and histopathological spectrum of adult gastrointestinal inflammatory neuropathy. Int J Surg Pathol 2021;29:246-56.  Back to cited text no. 3
    
4.
Henwood A. Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining. Biotech Histochem 2003;78:303-8.  Back to cited text no. 4
    

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Correspondence Address:
Suvradeep Mitra,
Department of Pathology and Lab Medicine, Academic Block, 2nd Floor, AIIMS, Bhubaneswar - 751 019, Odisha
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_539_21



    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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