Tissues Preparation in Histopathology

Preparation of Tissue (Different Methods of Preparation of Tissue)

• 2.1 Unfixed Tissue preparations

• 2.1.1 Imprint methods – Impression Smears of frozen section

• 2.1.2 Teased preparation

• 2.1.3 Squashed preparation

• 2.1.4 Frozen section

Author: Dr Pramila Singh

Preparation of Tissue (Different Methods of Preparation of Tissue)

The tissue preparation in histopathology involves a series of steps to process cells or tissues for microscopic examination. Tissue preparation develops a thin section of tissues to be fixed on slides and stained. It helps to visualize cellular components and tissue components under a microscope. This allows pathological studies and diagnosis of diseases. Tissue preparation processes require fixation, processing, clearing, embedding, sectioning, staining, and mounting of tissue sample.

2.1 Unfixed Tissue Preparations:

Unfixed tissue preparation in histopathology includes tissue sample handling and processing without undergoing the fixation step. The following steps are used in unfixed tissue preparation.

1. Immediate handling: Transfer the surgically removed tissue immediately to the medical laboratory. This prevents tissue degradation after its surgical removal from the body.

2. Trimming: Trim the tissues carefully to remove undesirable or excess materials. Trimming allows better handling of collected tissues and further processing steps.

3. Cryoprotection: Cryoprotoective agents such as sucrose or glycerol are used. Croprotective agents protect tissue from ice crystals that develop during the freezing of tissues. Cryoprotectives maintain the structural integrity of tissues during the freezing and sectioning stages.

4. Freezing: Freeze tissue immediately by using cryogens such as liquid nitrogen or any specialized freezing medium. Quick freezing preserves the cellular structure of tissues. This minimizes the formation of ice crystals.

5. Sectioning: Cut frozen tissues into thin sections using cryostat. Cryostat allows thin-section cutting at low temperatures. Carry out section cutting at -20 to – 30 degrees C. Collect thin sections on a clean sterile glass slide or on any other suitable surfaces.

6. Staining: Unfixed tissue sections are stained by using a suitable staining technique such as the frozen section staining technique or specialized staining technique.

7. Mounting: Mount stained sections using a suitable mounting medium and cover slide for microscopic examination.

Unfixed tissue preparation allows rapid evaluation and preservation of specific tissue components or molecular structures. But it has certain limitations over fixed tissue preparation such as reduced cellular preservation and alteration of cellular or tissue morphology.

2.1.1 Imprint methods – Impression Smears of frozen section:

It is a technique to analyze unfixed tissue preparation in frozen section analysis. Make impressions of the tissues directly on a clean and sterile glass slide to examine under the microscope. It allows quick evaluation of the tissues' characteristics without conventional tissue fixation and processing. It provides valuable information during intraoperative consultation. The following are the overview of the imprint method for impression smears of the frozen section:

1. Freezing: Freeze tissue immediately by using cryogens such as liquid nitrogen or any specialized freezing medium. Quick freezing preserves the cellular structure of tissues and minimize the formation of ice crystal.

2. Imprinting: Gently press clean sterile on the cut surface of a frozen specimen. Allow tissues to be in direct contact with a slide. This creates an imprint of the tissues cells and structure on the surface of the slide.

3. Staining: Fix the imprinted slide and stain using an appropriate staining solution. Hematoxylin and eosin stain (H & E Stain solution) or any other specific stains are used.

4. Mounting: Use a suitable mounting medium and coverslip to protect a smear.

5. Microscopic evaluation: Examine the stained imprint. Observe morphology, tissue architecture, specific features and abnormalities present in the smear.

2.1.2 Teased preparation under Unfixed Tissue preparations:

Teased preparation in impression smear is a specialized technique to study the morphology of individual cells without their fixation and staining. It allows quick evaluation of frozen tissue samples during surgical procedures and for preliminary diagnosis. The tissue sample is teased apart to separate individual cells. These cells are spread over a clean sterile slide to examine under the microscope.

But, this process does not provide clarity of cellular details as in formalin-fixed, paraffin-embedded tissue sections with H & E stains.

The following steps are followed in the teased preparation technique in the impression smear of frozen sections.

1. Tissue freezing: Freeze tissue samples rapidly using cryostat. Freezing tissue samples preserves cellular structures. This helps in visualizations of individual cells after the teasing process.

2. Sectioning: Use cryostat or microtome to get frozen sections of individual cells. Frozen sections of tissue samples are very thin (5 to 10 micrometers). Collect it carefully.

3. Impression smear: Press clean sterile glass slide gently onto frozen tissue section. This allows cells to adhere with sterile glass slide. Carefully lift the glass slide. This will create impression of frozen tissue section on sterile glass slide.

4. Teasing: Use needles or forceps on the slide to tease or scrap individual cells or clusters of cells. Teasing process separate the cells from tissues, preserve cells structure integrity.

5. Spreading and Drying: Carefully spread teased cells using forceps to form a monolayer of cells. Allow slides to dry in the air and its adhesion with the slide surface.

6. Microscope examination: Dried slide is ready for examination under the microscope. Additional staining solutions may be used to improve the visualization of cells.

2.1.3 Squashed Preparation under Unfixed Tissue Preparations:

Squashed preparation is a technique to examine unfixed tissue samples. It compresses tissues between two glass slides mechanically. This releases cells from tissues to form a single layer of cells to examine under the microscope. It is useful to study cell morphology such as cells' shape, size and their arrangement.

The following steps are followed in Squashed preparation under Unfixed Tissue Preparations:

1. Tissue collection: Collect tissues by biopsy or surgical excision. Handle tissue gently to preserve the structure of tissues and cells.

2. Tissue placement: Place the small piece of unfixed tissue on a clean sterile glass slide on the top of the tissue. Assure even distribution of tissue and avoid overlapping of cells.

3. Second slide placement: Place the second clean sterile slide on the top of the tissue. The second slide should completely overlap the first slide with

4. Compression: Press the upper second slide gently over tissues to form a thin single-cell layer film of cells. This causes spread of tissues and release of cells. Avoid too much pressure. This will distort morphology of cells and damage the cells.

5. Slide separation: Carefully and slowly lift second slide. Lifting process should not disrupt cells layer. Ensure cell adhesion to the first slide. Now, squashed preparation is ready.

6. Air drying: Allow squashed preparation to dry in air. Air drying helps the cells to adhere to glass slide.

7. Staining or microscopic examination: Stain squashed fry preparation by using suitable drying technique.

2.1.4 Frozen section under Unfixed Tissue Preparations:

Frozen section is a technique to prepare and examine unfixed tissue samples. It is a process to freeze and cut thin section of tissues. It is commonly used in surgical pathology during surgical process to examine tissues and diagnose disease. The following steps are followed in frozen section under unfixed tissue preparation.

1. Tissue collection: Collect tissues by biopsy or surgical excision. Handle tissue gently to preserve structure of tissues and cells.

2. Tissue freezing: Rapidly freeze the tissue using cryostat. Maintain temperature around -20 degrees C to -30 degrees C. This helps in tissue preservation and solidification.

3. Tissue embedding: Embed frozen tissue in a cryoprotective medium. Freeze the tissue with cryoprotective medium. This will form a frozen solid block.

4. Sectioning: Mount frozen tissue block onto cryostat. Cut it into thin slices. Thickness of frozen section shall be in between 5 to 10 micrometer.

5. Slide preparation: Coat a clean sterile glass slide with albumin or any charged molecules. This coating will enhance tissue adhesion on the slide. Carefully place the cut frozen section onto a clean sterile glass slide.

6. Staining or microscopic examination: Examine the cut frozen section under microscope. Frozen section may be stained using suitable staining solutions.

2.2 Fixed Tissue preparations (introduction only)

2.2.1 Paraffin embedding

2.2.2 Celloidin embedding

2.2.3 Gelatin embedding

Fixed Tissue preparations (introduction only):

Fixed tissue preparation is a process of biological specimens for microscopic examination. It preserves tissue samples obtained from patients in a fixed state. It helps to maintain the morphology of tissues and cells. It also prevents their decay and degradation. The following steps are followed in the fixed tissue preparations:

1. Tissue collection: Collect tissues by biopsy or surgical excision. Handle tissue gently to preserve structure of tissues and cells.

2. Fixation: Immediately deep the collected samples in a fixative solution such as formalin. Fixation helps to stabilize tissues by cross-linking cells protein. This preserves the cellular structure.

3. Fixative penetration: Fixative penetrates the tissue gradually to ensure even fixation. Allow sufficient time for fixation. Duration for complete fixation depends upon the type of cells and thickness of tissues/cells. Small biopsies require a few hours while larger specimens may require several days.

4. Gross examination: Examine tissue samples after fixation under the microscope. Microscopic examination assesses overall appearance, and abnormal masses in the tissues.

5. Tissue processing: Process the fixed tissue to remove water and replace water with a suitable medium.

2.2.1 Paraffin embedding under Fixed Tissue preparations:

Paraffin embedding is a technique to prepare fixed tissue samples for microscopic examination. It allows the preservation of tissue morphology and helps to cut thin and a high quality section of tissues. The following steps are followed for paraffin embedding under fixed tissue preparation:

1. Tissue fixation: Fix the freshly collected tissue by using suitable fixatives such as formalin.

2. Dehydration: Remove the water from the fixed tissues. Gradual dehydration is carried out by using alcohol in increasing the concentration such as ethanol 70%, ethanol 80%, ethanol 95%, and ethanol 100%. Alcohol replaces the water from the fixed tissues.

3. Clearing: Remove the alcohol from the dehydrated tissues by using clearing agents such as xyline, or any other organic solvents. The clearing step ensures proper permeation of the embedding medium. It also improves transparency to the tissue and their visualization of cellular structure under the microscope.

4. Infiltration: Now samples are ready for infiltration with the embedding medium. Infiltration involves dipping tissue samples into liquid paraffin. This allows the penetration of liquid paraffin into tissue spaces. This displaces the clearing agent from the tissue sample. Normally, paraffin is heated during this step to maintain its liquid state during the infiltration process. This ensures proper infiltration.

5. Embedding: Embed tissue sample in a mold with liquid paraffin wax. Mold helps the proper orientation for section cutting of the tissues. Paraffin is allowed to solidify to embed tissues.

2.2.2 Celloidin embedding under Fixed Tissue preparations:

Celloidin is an alternate embedding medium. It is used with alcohol and ether as an embedding medium for hard tissues. The following steps are followed:

1. Dehydration: Dehydrate the tissue sample by gradually replacing water with alcohol. Proper dehydration ensures proper tissue preservation.

2. Infiltration: Immerse dehydrated tissues in celloidin solution. Celloidin gradually enters tissues. Impregnate it with the embedding medium.

3. Embedding: Place the celloidin-impregnated tissues into a mold. Allow mold to dry, to form a hard and transparent block in which the tissue is embedded. 

2.2.3 Gelatin embedding under Fixed Tissue preparations:

Gelatin embedding is a technique to prepare fixed tissue samples for microscopic examination. It provides support and stability to tissues. The following steps are followed for gelatine embedding under fixed tissue preparation.

1. Tissue fixation: Fix the freshly collected tissue by using suitable fixatives such as formalin.

2. Dehydration: Remove the water from the fixed tissues. Gradual dehydration is carried out by using alcohol in increasing the concentration such as ethanol 70%, ethanol 80%, ethanol 95%, and ethanol 100%. Alcohol replaces the water from the fixed tissues.

3. Clearing: Remove the alcohol from the dehydrated tissues by using clearing agents such as Xyline or any other organic solvents. The clearing step ensures proper permeation of the embedding medium. It also improves transparency to the tissue and their visualization of cellular structure under the microscope.

4. Infiltration: Now samples are ready for infiltration with the embedding medium. Infiltration involves dipping tissue samples into liquid paraffin. This allows the penetration of liquid paraffin into tissue spaces. This displaces the clearing agent from the tissue sample. Normally, gelatin is heated during this step to maintain its liquid state during the infiltration process. This ensures proper infiltration.

5. Embedding: Embed tissue sample in a mold with liquid gelatin. Chill mold to allow gelatine to solidify. This will make a gelatine block. Mold helps there proper orientation for section cutting of the tissues. Paraffin is allowed to solidify to embed tissues.

   Ahuthor: Dr Pramila Singh