Histopathology
Table of Contents
Histopathology Introduction
Histopathology is the study of microscopic changes or abnormalities in tissue that are caused by a disease.
Tissue Processing And Staining
The most commonly used method of examining tissues microscopically is by section. Except for frozen sections of tissues, this requires the embedding of the tissues in a medium which will permit thin sections to be cut easily.
The embedding media used for all routine purposes is paraffin wax and the instrument used for cutting the sections for microscopy examination is called microtome.
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Paraffin Embedding Technique:
1. Fixation of Paraff Embedding:
- For good histological preparations, adequate and complete fixation of the tissue is important.
- Defects in fixation cannot be remedied at any later stage. It is essential that the tissue be fixed as soon as possible.
- Fixation is required to prevent putrefaction and autolysis, to preserve cell architecture.
- Tissues selected for sectioning should be fixed in the desired fixatives for 12 to 24 hours. The commonly used fixatives for routine laboratory procedures are formal saline.
2. Dehydration of Paraff Embedding:
- As paraffin does not mix with water, it is necessary to remove all traces of water before paraffin can permeate the tissue.
- This is achieved by immersion of tissues in increasing strengths of alcohol.
- For routine work, tissue is successively treated in 70%; 90%, and absolute alcohol (at least two changes) for 2-4 hours in each.
3. Clearing of Paraff Embedding:
- Since alcohol and wax are not miscible, the alcohol must be replaced by a wax solvent which at the same time clears the tissue and hence is known as the clearing agent.
- The most common agents in use are xylene (xylol), benzene, chloroform, and cedarwood oil.
- In routine procedures, tissue blocks after dehydration are immersed in Xylene for 1–2 hours with one change in half the time or till the tissue becomes transparent due to the permeation of xylene.
- Nowadays, automatic tissue processors are readily available for processing tissues.
4. Impregnation of Paraff Embedding:
- Through this process, the tissue is infiltrated with melted paraffin.
- The tissue on being cleared with xylene is transferred to a constant temperature paraffin bath of 58 to 60°C and is passed through at least two changes of paraffin of about 2–3 hours each.
5. Casting and Blocking of Paraff Embedding:
Tissue is now transferred from the final wax bath to a mold, filed with molten wax. The block is then quickly cooled and labeled.
Section Cutting:
- The paraffin block is trimmed and filed to a block holder.
- This is then cut into thin sections of 3 to 5 μ thickness by a special instrument called a microtome.
- Float sections in warm water at 45°C, prepare slides with a thin coating of equal parts of egg albumin and glycerin, and mount sections on the prepared slides.
- Water is now drained of and the slide is kept in an incubator before it is stained.
Staining of Paraff Sections:
- For general purposes, it is usual to employ some form of hematoxylin as a nuclear stain and one of the acid aniline dyes such as eosin for the cytoplasm.
- For deparaffinization heat the section in a hot air oven till paraffin melts. Dip the section in xylene 1–2 minutes and blot.
- Remove xylene with absolute alcohol and then 50% alcohol and finally wash in water.
Hematoxylin and Eosin (H and E) Stain:
- Stain section in hematoxylin for 30 minutes.
- Wash in tap water and keep in running tap water for 5 minutes till the color of the tissue is blue.
- Differentiate in acid alcohol (1% HCl in 70% alcohol) for a few seconds and note the light pink color of the tissue section.
- Wash in tap water and examine under the low power of a microscope to ensure proper differentiation and leave the section in running water for
- 5–10 minutes till it develops deep blue color.
- Counterstain in 1% aqueous eosin for 1–3 minutes.
- Wash in tap water and dehydrate in two changes of absolute alcohol for 1–2 minutes.
- Clear in xylene and mount in D.P.X.
Frozen Section Techniques
Frozen section diagnosis plays a vital role in the histopathology laboratory in intra-operative diagnosis.
Applications of Frozen Section:
- Rapid intraoperative diagnosis—to plan the ensuing surgery.
- To study the resection margins (malignant tumors) for completeness of excision.
- Stain for fat by Sudan 4, Sudan 3, Oil Red-O or osmic acid. (Fat is removed by xylene during paraffin embedding).
- Enzyme histochemistry: All enzymes are destroyed at temperatures above 56°C (required for paraffin embedding).
- Immunohistochemistry and immunofluorescence.
Disadvantages of Frozen Section:
- Serial sections cannot be obtained.
- Lack of embedding media causes distortion of structural details during cutting and handling.
- Unsatisfactory staining.
- Frozen sections can be obtained by using either a freezing microtome or cryostat. Nowadays cryostat is used.
Cryostat of Frozen Section:
- It is a refrigerated cabinet, in which a modified microtome is housed.
- All the controls of the microtome are operated outside the cabinet.
- It has an open top giving direct control over the cutting operation.
- The temperature remains constant with the top open or closed.
- They are either mechanically driven or hand-operated.
- The temperature of the cabinet is usually adjustable between 5° and 30°C. An operating temperature of 20°C is usually found to be suitable for most tissues.
- The tissue block, knife, and section are maintained at the same temperature.
Advantages of the cryostat over the freezing microtome:
- Thinner sections can be cut and the quality of the section is better in terms of staining.
- Serial sections can be cut.
Histopathology Slides
Acute appendicitis: It is one of the classical examples for acute inflammation.
- Gross:
- The appendix with mesoappendix is swollen due to the edema of the wall.
- Normal smooth, glistening, pale serosa is transformed into a dull, granular, red brown color due to engorgement (congestion) of the vessels and exudate over the serosa.
- Microscopy:
- The histologic criterion for the diagnosis of acute appendicitis is neutrophilic infiltration of the muscularis propria. Normally neutrophils can be seen in both mucosa and submucosa.
- Infiltration of the mucosa, submucosa, and muscularis propria (muscle layer) by neutrophils (acute inflammatory cells).
- Subserosal vessels are congested, and often there is a modest perivascular neutrophilic
infiltrate.
2. Pneumonia: It is also an example of acute inflammation. Pneumonia is inflammatory consolidation of the lung. In this condition, the air in the alveoli is replaced by inflmmatory exudate.
- Microscopy:
- Congested blood vessels in the alveolar wall.
- Alveolar spaces are packed with neutrophils and red cells.
- Proteinaceous flid (fluid exudates) with firing in the alveolar space.
3. Granulation tissue: It is called granulation tissue because of its pink granular appearance on the surface of a healing wound.
- Microscopy:
- Numerous newly formed capillaries in transverse or longitudinal sections.
- The abundant proliferation of fibroblasts.
- Infiltration by polymorphonuclear leukocytes, macrophages, lymphocytes and plasma cells.
4. Fatty liver:
- Gross:
- Specimen of a portion of the liver with a sharp border
- The cut surface and the outer surface is yellowish, and greasy.
- The capsule is stretched.
- Microscopy
- Structure of liver.
- Variable degree of vacuolation or clear spaces in the cytoplasm of hepatocytes.
Note: Intracellular accumulations of water or glycogen may also produce clear vacuoles.
- So, it is necessary to perform special techniques to distinguish these three types of clear vacuoles.
- Fat can be easily demonstrated by the frozen section method and these sections may be then stained by Sudan III (which gives orange color).
- Sudan 4 or Oil Red O, both impart orange red color.
5. Chronic venous congestion liver (nutmeg liver):
- Gross:
- The liver is enlarged.
- The cut surface shows a characteristic nutmeg appearance.
- The nutmeg appearance is due to alternate dark areas representing the congested center of each lobule, and light areas being the peripheral part with fatty change (steatosis).
- Microscopy:
- Structure of liver.
- The Central congested area of the liver lobule appears darker than the pale peripheral portions.
- Central hemorrhagic necrosis of the liver lobule with the disappearance of the liver cells in the center of the lobule.
6. Chronic venous congestion lung:
- Microscopy:
- Alveolar septa are widened due to the distension of capillaries with blood. The septa show a mild increase in the connective tissue.
- The alveoli are filled with macrophages containing hemosiderin pigment (heart failure cells).
- A special stain for the demonstration of Hemosiderin pigment is the Prussian blue reaction (Perl’s stain) which gives a blue color to hemosiderin pigment.
7. Actinomycosis:
- Microscopy:
- Central abscess contains bacterial colony (sulfur granule) characterized by radiating filaments (called ray fungus) surrounded by hyaline, eosinophilic, club-like ends which represent immunoglobulins.
- The periphery of the lesions shows chronic inflammatory cells, giant cells, and fibroblasts.
8. Rhinosporidiosis: It is caused by Rhinosporidium seekers. Usually, it occurs in the nasopharynx but may also be observed in the larynx and conjunctiva.
- Microscopy: Structure of nasal mucosa.
- A number of spherical sporangia (with chitinous wall) containing small basophilic round spores.
- Chronic inflammatory infiltrate in the subepithelial layer.
9. Tuberculosis of the lung:
- Gross:
- Microscopy:
- Structure of lung with multiple granulomas.
- Granulomas are composed of a central area of caseation surrounded by epithelioid cells, Langhan’s giant cells and lymphocytes.
10. Tuberculosis lymph node;
- Gross:
- Specimen of the lymph node.
- The cut surface shows map-like areas of cheesy yellow caseous necrosis.
- Microscopy:
- Structure of lymph node with multiple granulomas.
- Granulomas are composed of a central area of caseation surrounded by epithelioid cells, Langhan’s giant cells and lymphocytes.
11. Ameloblastoma:
- Microscopy: Follicular pattern is the most common pattern.
- The tumor shows follicles of varying sizes and shapes separated by fibrous tissue.
- The follicles consist of a central area of stellate cells.
- The peripheral layer of these follicles is cuboidal to columnar and appears to have a palisading arrangement.
12. Pleomorphic adenoma:
- Microscopy:
- Neoplastic cells show varying mixtures of epithelial tissue components intermingled with cells showing mesenchymal differentiation.
- The epithelial component consists of ductal cells and myoepithelial cells arranged in the form of ducts, acini, and irregular tubules.
- Mesenchymal-like elements show varying amounts of mesenchyme-like background of loose myxoid tissue, islands of hyaline, chondroid (cartilaginous), and mucoid matrix.
13. Tubular adenoma of the intestine:
- Microscopy:
- Consists of closely packed small rounded or tubular glands embedded in a stroma (increase in the number of glands and cells per unit area compared to the normal mucosa).
- The cells lining the glands are crowded and contain enlarged hyperchromatic nuclei. May show variable degrees of epithelial dysplasia.
14. Squamous papilloma:
- Microscopy:
- Papillary (figer-like) proliferation of the squamous epithelia of the epidermis with intact basement membrane and the basal cells.
- Central vascular connective tissue core.
15. Leiomyoma with hyaline change
- Microscopy:
- Interlacing bundles of smooth muscle fibers.
- Homogenous, glassy, pink material between the cells in many places.
16. Lipoma:
- Gross:
- Well-encapsulated mass.
- The cut surface shows yellow, and greasy, with fibrous septae.
- Microscopy:
- Fibrous capsule with septa containing blood vessels.
- Numerous fat cells are separated by fibrous septa.
17. Squamous cell carcinoma:
- Microscopy:
- Masses and groups of malignant squamous epithelial cells invading the dermis exhibiting pleomorphism, hyperchromasia, and mitotic.
- Epithelial pearls (concentrically laminated structures) in the midst of the invading masses of malignant cells.
- Invasion/infiltration of the underlying tissue.
18. Basal cell carcinoma (rodent ulcer) of skin:
- Microscopy:
- Groups and masses of darkly stained small epithelial cells (basaloid cells) with scanty cytoplasm invade the dermis from the basal layer of the epidermis.
- Palisading of the peripheral layer of cells of the epithelial cell masses.
19. Malignant melanoma of the skin:
- Gross:
- Microscopy:
- Structure of skin.
- Malignant cells laden with brownish-black pigment, grow down invading the dermis and subcutaneous fat.
20. Osteosarcoma:
- Gross:
- Microscopy:
- Tumors consist of cells of varying size and shape with large hyperchromatic nuclei and little cytoplasm.
- Formation of bone (osteoid matrix) by malignant cells.
- Mitotic and bizarre tumor giant cells are very frequent.
21. Giant cell tumor/osteoclastoma:
- Gross:
- Microscopy:
- A tumor is composed of uniform oval mononuclear cells with indistinct cell membranes arranged in a syncytium.
- Scattered within the background of such mononuclear cells are numerous uniformly placed multinucleated osteoclast-type of giant cells.
- These giant cells show 100 or more nuclei that are identical to those of mononuclear cells.
22. Metastatic (secondaries) carcinoma in the lymph node:
- Microscopy:
- Structure of the lymph node.
- Masses of malignant cells with nuclear pleomorphism and hyperchromasia.;
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