Tissue Processing
Tissue processing refers to the series of steps involved in preparing a biological tissue sample for microscopic examination. This process involves several steps, including fixation, dehydration, clearing, and embedding.
- Fixation: This is the first step in tissue processing, and it involves preserving the tissue’s structural integrity by using chemicals such as formalin. The fixative agent cross-links the proteins within the tissue, making it more resistant to decay.
- Dehydration: The next step involves removing water from the fixed tissue by soaking it in a series of alcohol solutions of increasing concentrations. This helps to prevent the tissue from swelling or shrinking during subsequent steps.
- Clearing: The third step involves replacing the alcohol with a substance that is transparent, such as xylene or other clearing agents. This allows light to pass through the tissue, making it easier to see under a microscope.
- Embedding: In this step, the tissue is placed in a mold and embedded in a medium, usually paraffin wax, which is then solidified. This helps to support the tissue and make it easier to cut into thin sections.
- Sectioning: The embedded tissue is sliced into thin sections using a microtome. The sections are placed on glass slides and stained with various dyes to highlight different parts of the tissue.
- Mounting: Once the sections have been stained, they are mounted on glass slides using a mounting medium, such as a synthetic resin, which is allowed to dry.
- Microscopic examination: The final step involves examining the tissue sections under a microscope to observe its structure and any abnormalities that may be present.
Tissue processing is a critical step in the diagnosis of many diseases, including cancer, and it requires precision and attention to detail to ensure accurate results.
Method of Tissue Processing
- Tissue Processing by Manual method
- Tissue Processing by Automatic Tissue Processor method
1. Tissue Processing by manual method
Introduction
The important steps of tissue processing by paraffin wax technique are dehydration, clearing, infiltration and embedding. After fixation and decalcification in the case of bone and calcified tissue, the tissue blocks are processed for the paraffin embedding, which give the tissue the necessary hardness for microtomy. The water is removed from the tissue by dehydration before, paraffin infiltration since paraffin is not miscible with water.
Requirements
- Cassette or capsules
- Ethyl alcohol, types
- 80% (v/v)
- 90% (v/v)
- 100% (v/v)
- 30% (v/v)
- 50% (v/v)
- Copper sulfate (anhydrous)
- Xylene
- Paraffin wax
- Embedding oven
- Lead pencil and pencil paper
- Embedding boxes (block’s, Leuckhard embedding boxes)
- Tissue
Procedure
Dehydration
Tissues thoroughly dehydrated before they are ready for embedding. In this procedure, water is completely removed from fixed tissues. This procedure is carried out as follows.
- Tissue blocks are placed in capsule or cassettes.
- The identification number is written with a lead pencil on a piece of paper.
- These tissue are passed through a series of increasing concentration of alcohol with the changes in the each concentration.
| Steps | Treatment | Time (hours) |
| 1-2 | 80% alcohol (2 times) | 1+1 |
| 3 | 90% alcohol | 1 |
| 4-6 | 100% alcohol (3 times) | 1+1+1 |
- The reliability of dehydration is checked by using in anhydrous copper sulfate (white). The last batch of alcohol is tested with anhydrous copper sulphate. If it turns blue, it indicate incomplete dehydration.
Clearing or Dealcoholization
Since xylene is miscible in both paraffin wax and alcohol. It is used as the clearing agent. It helps to replace alcohol and makes room for the paraffin during infiltration and impregnation. Refer to the following table.
| Steps | Treatment | Time (hours) |
| 7-8 | Xylene (2 times) | 1+1 |
Infiltration and Impregnation
During this procedure xylene is eliminated from the tissue by diffusion in the surrounding melted wax called infiltration. The wax afterward diffuse in the tissue by replacing xylene called impregnation. This procedure is carried out in the paraffin oven for 2 to 3 hours. The temperature paraffin oven should be between 50 degrees Celsius to 56 degrees Celsius.
| Steps | Treatment | Time (hours) |
| 9-10 | Paraffin (2 times) | 1+1 or 2 |
Embedding
Embedding means casting or blocking. In this procedure, the infiltrate and impregnated tissue is placed in warm liquid paraffin, which form a firm block after cooling. Embedding enables the tissue to be cut on a microtome. This procedure is carried out as follows.
- Leuckhard embedding box consists of 2 L shaped piece of heavy metals material brass is arranged on a glass plate.
- The specimen is placed at the bottom of the cavity. Orientation of the tissue is very important, and it is done according to the instruction given by the pathologist.
- The identification number either typed or written by a graphite pencil on a paper, should accompany the specimen identification number is placed adjacent to the tissue and care is taken so that it will not give in the way of the knife blade.
- Paraffin wax with high melting point 56 degrees Celsius to 58 degrees Celsius is first melted and filtered through a course filter paper. The filtered paraffin is then poured into the capacity of the box containing the specimen.
- The box (mould) is then placed in a container of cold water or kept in a refrigerator until the wax harden. It takes about 10 to 20 minutes.
- The hardened block is now ready for the section cutting on the microtome.
Tissue Processing by Automatic Tissue Processor method
Requirements
- Alcohol solutions of various concentrations such as 80%, 95%, 100% (absolute).
- Xylene
- Paraffin
- Automatic Tissue Processor (Histokinette).
- Tissue
Procedure
- Place the solutions and paraffin is respective beaker’s of the equipments.
- The timing lever is set at zero and the machine is started at 4.30 p.m. so that the tissue processing is complete during night hours.
- The basket with the cassettes automatically changes position and take bath in different reagents kept in the beakers in order to accomplish, dehydration, clearing, infiltration. The final dip is in the warm paraffin.
- Cassettes are opened next day morning for embedding.
Refer the following table for tissue processing.
| Steps | Timing | Solution | Periods |
| 1 | Starting point | 80% alcohol | 0 hour |
| 2 | 4.30 – 6.30 p.m. | 80% alcohol | 2 hours |
| 3 | 6.30 – 7.30 p.m. | 95% alcohol | 1 hour |
| 4 | 7.30 – 8.30 p.m. | 95% alcohol | 1 hour |
| 5 | 8.30 – 9.30 p.m. | 100% alcohol | 1 hour |
| 6 | 9.30 – 10.30 p.m. | 100% alcohol | 1 hour |
| 7 | 10.30 – 11.30 p.m. | 100% alcohol | 1 hour |
| 8 | 11.30 p.m. – 12.30 a.m. | Xylene | 1 hour |
| 9 | 12.30 – 2.30 a.m. | Xylene | 2 hours |
| 10 | 2.30 – 4.30 a.m. | Paraffin | 2 hours |
| 11 | 4.30 – 6.30 a.m. | Paraffin | 2 hours |
| 12 | 6.30 – 8.30 a.m. | Paraffin | 2 hours |