Optimization of PAS reaction and similar Schiff`s based methods for glycogen demonstration in liver tissue.

doi:10.21203/rs.3.rs-2591112/v1

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Optimization of PAS reaction and similar Schiff`s based methods for glycogen demonstration in liver tissue.

https://doi.org/10.21203/rs.3.rs-2591112/v1

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Purpose

Demonstration of glycogen in tissue has valuable diagnostic significance in several lesions, including certain tumors. Histochemical staining of glycogen with Schiff's dependent methods is affected by the type of fixative, the temperature of fixation, and oxidizing agents. This study aimed to evaluate different fixatives, temperatures of fixation, and oxidizing agents with variable durations of treatment to be used with Schiff's reagent for glycogen demonstration.

Methods

Archived paraffin blocks prepared from one rabbit's liver were used and 340 paraffin sections were stained with different oxidizers-Schiff methods.

Results

For tissues fixed at 4°C: 10% NBF, 80% alcohol, and Bouins's solution provided good staining results with PAS reaction. For tissues fixed at RT: both 10% NBF and 80% alcohol provided good staining results with PAS reaction. The other oxidizing agents provided poor results with all fixatives and temperature of fixation, with two exceptions of satisfactory staining results for 5% chromic acid for 10 minutes at both room temperature (RT) and 60°C.

Conclusion

Both 10% NBF and 80% alcohol seem to be the fixatives of choice for glycogen when the PAS reaction is used. However, Bouin's solution could provide good results at 4° C.

Oxidizing agent

PAS

glycogen

fixation

Glycogen is defined as a branched polymer of glucose that serves as a store of energy in the occasion of nutritional adequacy to be used in times of require [1]. Periodic acid Schiff (PAS) reaction was first reported for mucin demonstration [2]. It was used later to demonstrate some other substances, including glycogen. The action of periodic acid forms 3 Aldehydes on the 1,2 glycol linkage of carbohydrates found in tissue, and the resultant aldehydes can be colored by using Schiff's reagent [3]. However, these aldehydes can also be produced from polysaccharides by other oxidizers such as potassium permanganate and chromic acid, but they are stronger than periodic acid and they can destroy the produced aldehydes if adequate time is given [4, 5].

Fixation of glycogen seems to be a controversial issue; some workers suggest alcohol or picric acid-containing fixatives while others claim aqueous fixatives are adequate[6]. Type of fixative affects the appearance of glycogen [6]. Therefore, we included all these types of fixatives (aqueous, picric acid-containing solution, and alcoholic) and compared the staining results to determine the best combination for the demonstration of glycogen using PAS reaction and selected Schiff's reagent-based methods.

Study design

Archived paraffin blocks of known methods of fixation and processing were used in this study. Four fixatives were used: 10% NBF, Bouin's solution, Rossman's solution, and 80% alcohol, at both RT and 4°C. Three oxidizing agents were used: 5% chromic acid for 10, 20, 30, and 60 minutes at RT and 10 minutes at 60°C, 10% chromic acid for 20 and 30 minutes at RT, 2% FeCl3 for 5, 10, and 15 minutes, and 1% periodic acid for 5 minutes. The current study used known archived rabbit liver paraffin blocks with known methods of preparation. These archived blocks remained from another group at the department of histopathology and cytology at the faculty of medical laboratory sciences–University of Khartoum, Sudan, and the archived blocks were prepared from the rabbit:

Sampling

The rabbit’s liver was rinsed with normal saline, sliced into 8 pieces approximately 5×3×3 mm, and transmitted to the appropriate fixative.

Fixation

Each piece was fixed in one of four fixatives: 80% alcohol, 10% NBF, Bouin’s and Rossman’s solution at RT and 4°C in a refrigerator for 24 h. Each piece was placed in a labeled cassette according to the fixative type and fixation temperature. Subsequently, they were transferred to the processing machine.

Post-fixation Treatment

Following Rossman's solution, samples were moved to 95% alcohol for 2 h, and after Bouin's fixation they were treated with 75% alcohol for 2 h [7]. Subsequently, samples were transferred to labeled cassettes according to the type and temperature of fixation.

Tissue Processing

For dehydration, 80% alcohol fixed samples were treated with one change of 80% alcohol and 4 changes of absolute alcohol for 1 h each. Bouin’s solution fixed samples were dehydrated in one change of 75% and 90% alcohol, then four changes of absolute alcohol 1 h each. Rossman’s solution fixed samples treated in one change of 95% alcohol, then by four changes of absolute alcohol 1 h each. 10% NBF fixed samples treated with one change of 70% and 90% alcohol, then by 4 changes of absolute alcohol 1 h each.

For clearing, all samples were treated with two changes of xylene 2 h each. For impregnation, samples were treated with two changes of melted paraffin wax for 2 h each. For embedding, samples were embedded in paraffin wax using metal molds. Finally, they were left to harden at RT, then in a refrigerator.

Sectioning

340 labeled slides were prepared and 4 µm sections were cut by a rotary microtome, 11 sections from each block (3 tests and 1 control for each variable).

Staining

All sections were treated with 2 changes of xylene for 3 minutes each, absolute alcohol, 90% alcohol, 70% alcohol, and distilled water for 2 minutes each. Subsequently, they were stained using the following techniques.

Regarding the PAS reaction, sections were placed in1% periodic acid for five minutes, rinsed in distilled water (DW), treated with Schiff's reagent for 15 minutes, washed in running tap water for 10 minutes, stained with Harris's hematoxylin for 3 minutes, differentiated in 1% acid alcohol, blued in running tap water for 10 minutes, dehydrated in absolute ethanol, cleared in xylene and mounted in D.P.X [8].

For the chromic acid-Schiff method, sections were treated with 5% chromic acid for 10, 20, and 30 minutes, 1 hour at RT, 10 minutes at 60o C, and 10% chromic acid for 20 and 30 min. Sections washed well in three changes of DW, treated with Schiff's reagent for 15 minutes, washed in running tap water for 10 minutes, stained with Harris's hematoxylin for 3 minutes, differentiated in 1% acid alcohol, blued in running tap water for 10 minutes, dehydrated in absolute ethanol, cleared in xylene and mounted in D.P.X [9].

For the Ferric chloride-Schiff reaction, sections were treated with 2% ferric chloride acid for 5, 10, and 15 minutes, washed well in DW, treated with Schiff's reagent for 15 minutes, washed in running tap water for 10 minutes, stained with Harris's hematoxylin for 3 minutes, differentiated in 1% acid alcohol, blued in running tap water for 10 minutes, dehydrated in absolute ethanol, cleared in xylene and mounted in D.P.X [8].

For negative controls, sections were treated with saliva for1hour at 37°C and washed in tap and distilled water, then stained according to the requested technique [10].

Scoring System

Two examiners did a subjective evaluation of the staining results. Slides with technical errors that render them difficult to be evaluated were excluded, and the average of the scores was calculated for each test slide. Finally, the quality of glycogen staining results was written as follows:

Poor: 0 - < 5: Weakly stained glycogen, granules are small and indistinct, and recognizing glycogen is difficult.

Satisfactory: 5 - < 7: Visible glycogen but stained faintly.

Good: 7- < 9: Glycogen is fairly distributed evenly throughout the stained section and visible.

Excellent: 9–10: Visible glycogen which has well-stained large granules which are fairly evenly distributed throughout the stained section.

Ethics Approval

We used archived paraffin blocks in this study. Department of histopathology and Cytology, Faculty of Medical Laboratory Sciences, University of Khartoum, accepted the study and no ethical approval was required.

Both 10% NBF and 80% alcohol appear to be the fixatives of choice for PAS reaction compared to the other two fixatives. In which, they provided good staining results. However, Bouin's solution provided good results at 4°C (Table 1 and Fig. 1).

For Bouin's solution at 4°C, the highest average score of staining quality was detected with 1% periodic acid for 5 min which scored 7.7 (good) results (Table 1 and Fig. 2).

For Rossman’s solution at both 4°C and RT, the highest average score of staining quality was detected with 1% periodic acid for 5 min, which provided satisfactory staining results (Table 1 and Fig. 3).

For 10% NBF at both RT and 4°C, the highest average score of staining quality was detected with 1% periodic acid for 5 minutes, which scored. 8.7 (good) for each (Table 1 and Figs. 4, 5, 6, and 7).

Similarly, periodic acid provided the highest score for 80% alcohol at both 4°C and RT, which scored 8 and 7.7 (good) respectively (Table 1 and Figs. 8, 9, 10, and 11).

Interestingly, 5% chromic acid-Schiff reaction at both RT and 60°C for 10 min provided satisfactory staining results with 80% alcohol at 4°C (Table 1 and Fig. 12). However, 10% chromic acid at RT for 20 and 30 minutes provided poor staining results with all fixatives (Table 1). 2% FeCl3 for 5, 10, and 15 min provided poor staining results with all fixatives and temperatures of fixation (Table 1 and Fig. 13).

Table 1

Average scores of staining quality for different oxidizing agents – Schiff reactions following different fixatives and temperatures of fixation.
Oxidizing agent, concentration, and duration.	Fixatives & temperatures of fixation
Oxidizing agent, concentration, and duration.	Bouin’s solution		Rossman’s solution		10% NBF		80% alcohol		Total And Average
	RT	4^°C	RT	4^°C	RT	4^°C	RT	4^°C	Total And Average
1% Periodic acid, 5 min	4.6* poor	7.7 good	5.6** satisfactory	6.5* satisfactory	8.7 Good	8.7 Good	7.7 Good	8 Good	57.5 (7.2)
5% chromic acid at RT, 10 min	4.2 poor	4.5 poor	1.33 poor	3.1 Poor	3.3 poor	3.7 poor	1.8 poor	5 satisfactory	26.9 (3.4)
5% chromic acid at RT, 20 min	3 poor	2.2 poor	0.5 poor	1.2 Poor	2.8 poor	4.2 poor	1 poor	3.7 poor	18.6 (2.3)
5% chromic acid at RT, 30 min	2.83 poor	1.3 poor	0,7 poor	0,83 poor	3.2 poor	3.3 poor	0.5 poor	3.3 poor	15.7 (1.9)
5% chromic acid at RT, 1h	2.6 poor	0.83 poor	0.5 poor	0.8 poor	1 poor	3 poor	0.5 poor	0.2 poor	9.4 (1.1)
5% chromic acid at 60⁰C 10 min	3.8 poor	3 poor	0.7 poor	0.5 poor	3.7 poor	3.2 poor	2.7 poor	5.7 satisfactory	23.3 (2.9)
10% chromic acid at RT, 20 min	2.3 poor	0.7 poor	1.2 poor	0.83 Poor	0.7 poor	2.3 poor	0.3 poor	0.2 poor	8.5 ( 1.0)
10% chromic acid at RT, 30 min	1.2 poor	0.7 poor	0.83 poor	0.5 poor	0.7 poor	1.2 poor	0.2 poor	1 poor	6.3 (0.7)
2% FeCl_{3 ,} 5min	0.7 poor	0.5 poor	0.5 poor	***	0.3 poor	0 poor	0.3 poor	0.3 poor	2.6 (0.37)
2% FeCl_{3 ,}10min	1.7 poor	0.7 poor	0.2 poor	***	0.2 poor	0.2 poor	0 poor	0 poor	2.6 (0.37)
2% FeCl_{3 ,} 15min	0.7 poor	0.3 poor	0.33 poor	***	0 poor	0.5 poor	0.2 poor	0.5 poor	2.5 (0.36)

*The mean was calculated from 3 slides (3 slides were excluded).

**The mean was calculated from 5 slides (1 slide excluded).

***Excluded.

The quality of glycogen staining could be affected by the fixative in use and the temperature of fixation, as well as using an appropriate oxidizing agent to be used with Schiff's solution. In this study, we noticed that both 10% NBF and 80% alcohol are the best fixatives for PAS reaction. In which, gave good staining results at both RT and 4°C. However, Bouin's solution provided good staining results when fixation is carried out at 4°C. These findings support the results got by Kugler and Wilkinson (1964), who reported that ice-cold 80% alcohol preserves 81% of the glycogen found in the tissue in different histochemical reactions [11]. the good staining results after 10% NBF fixation can be explained by the action of formalin which fixes proteins that are associated with glycogen [6].

In our results, we found periodic acid to be the best oxidizing agent when compared to the other evaluated oxidizers in the current study. This finding agrees with what is found in literature where periodic acid appears to be the most commonly used oxidizing agent to be used with Schiff’s reagent for glycogen demonstration [12, 13, 14].

Chromic acid is a strong oxidizing agent, which could oxidize numerous groups beyond the reactive aldehyde stage [9]. In addition, Graf and Klessen (1981) reported that glycogen cannot be easily detected using chromic acid Schiff’s technique [15]. This could be attributed to the fact that chromic acid is a strong oxidizing agent as it can destroy the produced aldehyde [4]. Our findings are in concordance with this information. However, we found that the quality of the chromic acid-Schiff method for glycogen demonstration depends on fixative, temperature, oxidizing agent concentration, and duration of treatment. It can yield satisfactory results when the above-mentioned factors are considered. This was clear with a 5% chromic acid-Schiff reaction at both RT and 60oC for 10 min, where satisfactory staining results were got with 80% alcohol at 4°C.

At the level of the current study, both 10% NBF and 80% alcohol seem to be the best fixatives for glycogen demonstration using PAS reaction, followed by Bouin’s solution at 4°C. Periodic acid gave the best results in comparison with other oxidizing agents used in this study.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

- Marwah Abdelrahman Abdellah, Masia Aldai Abdallah, Abeer Babiker Abdalrheem, Alobeid Mohammed Gubara: Technical work including sample preparation, fixation, processing, cutting, and staining.

- Samah Abdelrahim Batran: Assessment of results and revising the manuscript.

- Yosef Mohamed-Azzam Zakout: Planning, designing, and directing the study, writing and preparing the manuscript, and assessment of results.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

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No competing interests reported.

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Version 1

posted

You are reading this latest preprint version

Optimization of PAS reaction and similar Schiff`s based methods for glycogen demonstration in liver tissue.

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Methods

Study design

Sampling

Fixation

Post-fixation Treatment

Tissue Processing

Sectioning

Staining

Scoring System

Ethics Approval

Results

Discussion

Declarations

References

Additional Declarations

Status:

Version 1