Special Stains: Enhancing Tissue Diagnosis Beyond H&E

Abstract

The examination of tissues to identify the structure, and composition and provides diagnosis where necessary largely entail staining the tissues before conducting the observation. This report sought to evaluate the application of special stains in haematology experiments since most tissues require the application of these stains for clear, precise and conclusive observation. In this report, periodic acid Schiff stain method was applied to liver tissues of the lamb to identify the key histological features when observed under the microscope. The experiment revealed that the nucleus was blue in color and the color of the cytoplasm was magenta. This study helped reveal clearly, under magnification the key features of the liver tissue and determine the health of the liver tissue under observation since the resulting color depends on the presence of the polysaccharides and glycoproteins which are identified by the periodic acid Schiff staining experiment.

Introduction

In hematology laboratories, most tissues would be stained with haematoxylin and eosin (H&E) at least initially and help pathologists arrive at a diagnosis. However, in many situations, H&E alone is not sufficient and should be supplemented with special stains for precise tissue diagnosis. The special stain is a term for all other forms of stains other than H&E that can be used since most tissues, especially liver tissues routinely required these special stains. PAS is an example of the commonly used special stains.

Periodic Acid Schiff (PAS) staining is a staining method used to identify polysaccharides and mucosubstances such as glycoproteins and glycolipids in tissues. In human an animal tissue, the main polysaccharide identified by PAS staining is glycogen which is present in tissues such as skeletal muscles, kidney, and liver. PAS is also used in tissue basement membranes which are thin layers that anchor and support epithelium and endothelium to underlying connective tissue (Hauser, 2005).

PAS staining methods can be used in different diagnostic circumstances. The staining patterns of lymphocytes which can be identified using PAS staining are helpful in making therapeutic decisions in cases of lymphocytic leukemia. The diastase (α-amylase) digestion procedure, accompanied by PAS staining can aid in the diagnosis of glycogen storage disease. PAS staining can also be useful in the demonstration of fungal organisms in the tissue section.

The reactivity of the PAS technique depends on the monosaccharide units in tissues. The first reaction of the stain involves periodic acid acting as an oxidizing agent, oxidizing carbon-to-carbon bonds between two adjacent hydroxyl groups (Carson et al, 2009). This produces aldehyde groups which are Schiff reactive. In the second reaction, Schiff reagent which comprises of the mixture of basic fuchsin, hydrochloric acid, and sodium metabisulphite reacts with the formed aldehyde groups in the tissues to produce a bright magenta color. The intensity of the color depends on the concentration of the hydroxyl groups originally present in the monosaccharide. Diastase, on the other hand, hydrolyzes starch, glycogen and breaks down products originating in the tissue polysaccharides. The slide of tissue containing distance, when compared on the glycogen slide will not reveal PAS stain.

The liver was our main tissue of study. The liver lobules are the functional and structural units of the liver. The lobule is surrounded by 6 portal veins and centered on Centro lobular vein which is a terminal twig of the hepatic vein. The portal veins result into the portal tracts with a series of branches formed between adjacent portal tracts which generate sinusoids that drain blood towards the center of the lobule

Materials, experimental outline and rationale

To conduct out the PAS staining test, there are materials that were used. The tissue used for the study was paraffin embedded lamb liver sections at 4-5 μm. Solutions and reagents were also used. They were: α-Amylase/diastase, 0.5% periodic acid solution, Schiff reagent, and Mayer’s Haematoxylin solution. Xylene and ethanol were also used.

Prior to the practicals in the hood, the sections were deparaffinized in 2 changes of Xylene for 5 minutes each. The sections were then rehydrated in 2 changes of absolute alcohol, 3 minutes each. The sections were further rehydrated in 90% of alcohol for 3 minutes and 70% of alcohol for 3 minutes. The sections were then brought to distilled water

For the selected sections that needed amylase digestion, they were treated with amylase solution for 20 minutes and then washed in running water. The slide was then washed with dH2O for 30 seconds and then excess dH2O was tapped off before applying 0.5% periodic acid on the lines and stained them for 5 minutes. The excess of the solutions was then tapped off on the tissue paper and the slides were transferred to a Coplin Jar and rinsed for 3 changes of dH2O, 1 minute each.

The slides were placed back in the tray and excess water was wiped off. Schiff reagent was added on the slides and observed for color change. Schiff solution was then tapped off and the slide placed back in the Coplin Jar with warm tap water for 2 minutes and changed to fresh water after 1 minute. The slides were placed back in the tray and excess water was wiped off then haematoxylin applied for 8 minutes for counterstaining. Thereafter, haematoxylin was tapped off before washing the slides in the Coplin Jar in running water for another 3 minutes.

In the hood, the sections were dehydrated through 70% of alcohol for 2 minutes and 90% of alcohol for 2 minutes and 2 changes of absolute alcohol 2 minutes each. The sections were then cleared in 2 changes of xylene for 2 minutes each. The slides were then mounted with xylene-based mounting medium and a coverslip. Finally, the sections were observed under a light microscope, images captured and the structure of the tissue observed.

The procedure referred from:

Chapter 8 (Histochemistry: the general considerations and histochemistry of carbohydrates) in Cellular Pathology, by D, J Cook 3rd Edition.

Results and findings

After conducting the procedures, the result of the observation was recorded. The nuclei were blue in color. The glycogen in the cytoplasm was deep red (magenta) in color. On the digested slides, there was no magenta color in hepatocytes. The photos from the findings are presented below.

The histological features of the sections were also identified when the slides were observed under the magnified image of the microscope. The artefact, euchromatic bi-nuclei, and hepatic artery were observed. Other features observed were the nucleus which was seen in blue color and the connective tissues surrounding the hepatic artery. The glycogen stores were also identified by the magenta color

Discussion and conclusion

Studies have found out that PAS is used for identifying glycogen in tissues such as the liver but removing glycogen with diastase enhances detection of nondigestible materials (Bancroft and Gamble 2002). These include alpha-1 antitrypsin globules, basement membranes, and fungal organisms. In patients with alpha-1-antitrypsin deficiency, accumulation is manifested with bright magenta globules (Cook and Warren 2015). There is also larger and more numerous globules in tissues with the homozygous disease. This helps to ascertain whether the findings of the practicals could identify the presence of either condition in the tissues. Figure 1 presents tissues not treated with diastase while figure 2 presents sections subjected to diastase. The PAS staining practical positively identifies glycogen since the color of the sections conform with the other studies.

In conclusion, PAS staining practical can be useful for making the diagnosis of tissues. Tissues treated with PAS as shown in the findings and literature will identify glycogen in Magenta color. Combined with diastase, nondigestible materials can be revealed. This can help identify diseases in tissues. However, the accuracy of the results depends on carefully following the procedures for conducting the experiment.

References

• Bancroft J and Gamble M. 2002. Theory and practice of histological techniques. 5th edition. London. Churchill Livingstone.
• Carson, Freida L.; Hladik, Christa 2009. Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press.
• Cook D J & Warren PJ. 2015. Cellular Pathology. An introduction to techniques and application. 3rd Edition. Scion Publishing Ltd.
• Hauser C. 2005. Mayo Clinic Gastroenterology and Hepatology Board Review. CRC Press.