Report On Histopathological And Histochemical Techniques

Discuss about the Report for Histopathological and Histochemical Techniques.

The aim of this exercise is to demonstrate the dissection and preparation of tissue sections that were taken from a rat for histopathological analysis. Furthermore, difficulties and issues occurred during the conduct of the experiment are discussed. Additionally, histological features of the rat tissue by using Haematoxylin and Eosin (H&E) and different special stains are also discussed (1-2).

Initially, specific organs from the rat were ordered to be dissected. These organs include liver, kidney, lung, trachea, duodenum and femur. In order to obtain these organs from the rat, the rat was dissected followed by cutting of specific organs into an appropriate size and placed in cassette and finally fixed with a fixative agent like 10% normal buffered formalin. The fixation process was crucial because it prevented the tissue from autolysis, putrefaction, osmotic swelling, shrinkage and loss of cellular component (2-4).

Tissue processing was performed by the pathology technician and Leica ASP200s tissue processor was used for tissue processing. Another key point to mention here is that, unlike other tissues, femur tissue required decalcification prior tissue processing. The next step was embedding which plays a critical role in improving the stability of tissue for long time and supporting the tissue section. The tissues were placed centrally and correctly orientated in mould followed by adding paraffin wax and then were placed in cold bench for solidification (1, 5).

Furthermore, sectioning of tissues was performed by setting the solidified block in the microtome. Trimming was done at 10 µm till the full-face of the tissue was exposed and followed by cut at 4 µm to get ribbons. There are many aspects that should be considered to get a good quality sections like cooling the block, use of sharp blade and optmum water-bath temperature. After the complete drying up of the slides, the staining step took place. Progressive Haematoxylin and Eosin (H&E) was used to demonstrate the details at nuclear level and cytoplasmic features of the tissues. Next step was to perform staining with different special stains. Hall, Perls and Congo red stains were used to demonstrate bilirubin, haemosiderin andamyloid, respectively in liver tissue. Periodic Acid Schiff’s (PAS) along with Diastase Digestion were used to demonstrate carbohydrates like glycogen in kidney. Alcian Blue Acid Schiff’s stain (AB-PAS) was also used to demonstrate and differentiate different types of acid mucin in duodenum. Moreover, to evaluate fibrosis of esophagus and trachea, Masson’s Trichrome (MT) was used. Finally, Verhoeff’s Elastic stain (VVG) and Gomori’s Aldehyde Fuchsin stain (GAF) were used to demonstrate elastic fibres (6-8).

Liver

Liver:

The liver was bathed with blood which made some difficulties in removing it. After that it was cut into 10% normal buffered formalin. Afterward, it was cut into small pieces of around 8*7 mm and placed in a cassette. Tissue processing was performed by pathology technician. There was no issue in embedding step. However, there was difficulty during sectioning procedure which involves dissolving and also, possibility of breakdown of the tissue when it was placed in the water-bath. A couple of techniques were used to resolve this issue like cooling the block and changing the blade, however all these efforts failed. Even Janine tried one time but it was the same result which is dissolving of the tissue in water-bath. Eventually, Janine said this issue might be caused by bad tissue processing. Although the tissue was damaged, five slides were prepared as requested. Progressive H&E method was used to demonstrate the features of the tissue, which reports initial diagnosis. However, as a result of bad processing and damaged tissue, it was difficult to recognise the liver’s architectures in the H&E slide. Moreover, three special stains were requested to be performed. Congo red for amyloid, Perls for haemosiderin and Hall’s for bilirubin was used. Five slides were performed, two for Perls, two for Hall’s and one for Congo red. Perls stained positively in the control slide by illustrating the haemosiderin with dark blue colour but there was no sign of haemosiderin in the rat liver. The control slide of Hall’s stain illustrated the bilirubin with olive green colour but the test slide was negative for bilirubin. There was only one slide for Congo red which was control slide and it demonstrated the amyloid with red in colour. However, the tested slides should be repeated because the tissue was damaged and most of the liver’s structure was disappeared (5-9).   

Kidney:

Dissection of the kidney was simple because it is not attached to any other organ. Since, the kidney was cut in half, there was no issue in the embedding step. The cut side of the kidney was placed at the bottom of a cassette. However, sectioning kidney tissue was challenging because there was a problem in tissue processing as Janine explained in the class. Although there was poor processing and improper sectioning of kidney tissue, progressive H&E method was used to demonstrate the histological features of kidney. In H&E slides numerous glomeruli were observed in the cortex and the bowmen’s capsule of glomerulus. It also showed the inner and outer medulla, distal tubules and collecting tubules. Moreover, two control slides of kidney tissue were given to perform Periodic Acid Schiff (PAS) and (PAS-Diastase) stains. PAS stain demonstrated different types of carbohydrates such as glycogen and natural mucin in the tissue. However, it was difficult to distinguish glycogen from PAS positive mucins; therefore, PASD was used to remove the glycogen. PASD slide were treated with fresh human saliva, which performed function of diastase agent. The results demonstrated that the glycogen was present in PAS slide with magenta colour whereas no magenta colour was present in PASD slide. Rat kidney stained positively by exhibiting the magenta colour in glomeruli and tubules. It also showed thickness of the basement membrane of the glomeruli (6-10). 

Kidney

Lung:

The dissection of the lung was challenging because it is located under the rib cage of the rat. Thus, a scissor was used to facilitate removal of the lung. There were no issues in the embedding step. The sectioning step was difficult and that was due to inadequate processing. However, sectioning of the lung was easy as compared to the liver and the kidney sectioning. Progressive H&E method was used to demonstrate histological features of lung and to report initially diagnosis of the lung tissue. Although there was poor processing for lung, the lung’s features were demonstrated such asbronchioles, alveolar & alveolar ducts in the H&E stained slides (6-10).

Duodenum:

Detaching the duodenum was difficult because it was hardly visible, as it was bathed with blood. Two sections were cut for duodenum, long section from the edge and small section from the middle, as requested from the administrators. There were some difficulties in embedding the small section due to its small size, but it was managed by using two forceps. The sectioning step went well and smoothly as a result it gave neat and clean ribbons. Progressive H&E method was used for staining, which demonstrated different structure of the duodenum. Structures observed in the duodenum were mucosa, submucosa, goblet cell and smooth muscle. In addition, Alcian Blue/PAS stain was performed in two slides like control slide and duodenum slide and it demonstrated presence of mucin. Both slides demonstrated neutral mucin which was represented with magenta colour while acid mucin was negative (6-11).

Oesophagus & Trachea:

There were some difficulties in detaching oesophagus & trachea because these tissues were located under the rib cage of the rat. Embedding step was challenging due to the small size of one of the sections obtained from these tissues. So two forceps were used to fix the tissue while embedding. Eventually, it embedded in a right position. As a result, sectioning by the microtome went very well and easy which gave nice ribbons. Like other tissues progressive H&E method was used for oesophagus & trachea staining. In H&E slide, different structures were demonstrated like mucosa and submucosa layers of the trachea, also thyroid glands were seen. However, it was difficult to see the folds of the oesophagus. Furthermore, Masson’s Trichrome was used to demonstrate fibrosis that was present in these tissues. The control slide showed collagen and it was differentiated with muscle collagen. The tested tissues showed presence of normal level of collagen which reflects these tissues were normal (5-9).

Lung

Bone (femur):

One of the hardest organs to detach was bone, so scalpel was used to cut the femur from the leg. In addition, it should be free of any attached muscle, so it was removed by scalpel. Not like any other tissues, femurs had important step in tissue processing which was decalcification. The next step was embedding which was easy because the hardness of femur and it helped in fixing the tissue at the time of embedding. There were no issues in sectioning and as result clean ribbons were obtained. Progressive H&E method was used to stain different features of the femur. It demonstrated collagen fibres, bone marrow & cartilage (5-9; 13).

Conclusion:  

The purpose of the current study was to demonstrate the method of dissection, tissue processing, preparation and staining of slides for histological observations of collected tissues. In order to obtain a high quality sections, all precautions were taken at each step right from the dissection until the mounting of tissues. In addition, this exercise taught the importance of tissue processing because it could affect the section quality. Moreover, it explained the correlation between H&E and special stains in microscopic examination. Finally, it was understood that accurate diagnosis of histological sections could be obtained from accurate histological preparations.

1-Write up a processing schedule for an urgent endoscopic biopsy eg. Duodenum?

Solution	Time Required	Vacuum	Temperature (C°)
Formalin (10%)	10 Minutes	Yes	38°C
Formalin (10%)	10 Minutes	Yes	38°C
Alcohol (70%)	10 Minutes	Yes	38°C
Alcohol (95%)	10 Minutes	Yes	38°C
Alcohol (95%)	10 Minutes	Yes	38°C
Absolute Alcohol	10 Minutes	Yes	38°C
Absolute Alcohol	10 Minutes	Yes	38°C
Xylene	10 Minutes	Yes	38°C
Xylene	10 Minutes	Yes	38°C
Paraffin Wax	10 Minutes	Yes	38°C
Paraffin Wax	10 Minutes	Yes	58°C

(1, 2)

 2- Please list the different types of fixation available?

There are two methods available for fixation, physical and chemical methods.

Physical fixation method include:

• Heating (rarely used).
• Micro-waving (widely used)
• Cryo-preservation (that for freeze drying).

Chemical fixation method  include:

• Mercuric chloride.
• Osmium tetroxide.
• Picric acid.
• Alcohol both (Ethanol and Methanal).
• Acetic acid.(4, 12)

3-What are the other clearing agents that could have been used for processing your specimens? Briefly discuss their advantages and disadvantages?

Clearing agents	Advantage	Disadvantage
Xylene	Commonly used because of it is less harmful nature.	It is harsh and may cause solidification and brittleness to the tissue.
Chloroform	Not as harsh as xylene. Not harmful to the tissue,  used in the experiment.	Slower than xylene. It may cause health problems for the technician.
Limonene-derivatives	Unlike xylene, limonene derivatives have low toxicity levels.	It has a pungent odor.
Cedar wood oil	It is characterized by gradual action and lacks significant effect on hardening of tissues.	Expensive. Slow.

(1-2; 10)

4-What precautions should be taken when setting and loading a tissue processor?

• Make sure that all reagents are clean.
• Adjustment of the processor depends on type of tissue because bone tissues needs decalcification prior processing.
• Well trained staff should run the machine.
• Safety precautions should be followed (1, 10)
  

5-What effects can prolonged decalcification have on tissues?

Prolonged decalcification causes tissue damage. Moreover, it may spoil the staining of basophilic structure like cell nuclei and causes weakness of the softer tissue elements, especially when strong acid decalcifying agent is used. In addition, it destroys the calcium that is present in the bone and eliminate the morphology of the cell (1-2; 13).

6- What methods other than a chemical test for calcium ions, are available for determining the end point of decalcification? Comment on their reliability.

• Radiography: sensitive method but it may lead to tissue damage due to X-ray exposure.
• Calcium oxalate.
• Physical method: but it may lead to tissue artefact if the tissue squeezed hardly (1-2; 13).
  

7-When embedding tissues, what should be avoided in order to ensure that quality is achieved?

• Paraffin wax and the cold plate must be in a right temperature.
• Choosing an appropriate size of moulds is critical in order to achieve high quality sections.
• Clean forceps should be used.
• Tissue should be well orientated (1, 10).

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