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Tissue processing is a critical step for preparing samples for histopathological analysis. Getting the specimen right before processing is a crucial factor as as fresh specimens are delicate, and mishandling can potentially lead to sample loss. A typical room temperature tissue processing of 3 mm sample requires approximately 12 hours (Fixation: 10% formalin (120 min), alcoholic formalin (60 min, 60 min); Dehydration: 95% alcohol (60 min, 45 min), absolute alcohol (60 min); Clearing: xylene 60 min, 60 min; Infiltration: paraffin (30 min, 60 min, 90 min)).

Fixation for stabilizing proteins and hardening the tissue

The samples once procured should be immediately fixed if possible, or as soon as they are received after being transported. Key factors to consider during fixation include temperature, size of storage container, volume ratio of the fixative, pH, salt concentration, and incubation time. The choice of fixing solution depends on the detected antigen. Acetone and alcohol are used when precipitating fats and sugars and for maintaining immunologic competence;aldehydes (formalin [10% neutral buffered], 4% paraformaldehyde) are commonly used due to strong penetrability and less contractibility and background; and non-aldehydes are used in tissue fixation of peptide hormones.

Formaldehyde fixation is commonly performed at room temperature, and adequate volume of fixative in minimum1:20ratio for 3–4 mm thickness should be used in a container of appropriate size to avoid distortion of the fresh specimen and ensure good quality fixation.Isotonic buffers of pH 7.2–7.4 are recommended to avoid tissue swelling or shrinkage, and maintain ultrastructure with minimal distortion, and the duration of fixative exposure should be optimized for every specimen type.

Dehydration for removing unbound water

The size and penetrability of the tissue determine the time required for this step.Insufficient dehydration of the sample can occur due to carry over of fixative into the processing alcohol. The presence of water in absolute alcohol stations before the clearing stations can also lead to incomplete dehydration. This residual water gets trapped during paraffin infiltration, resulting in soft samples that are difficult to cut in in microtomy. Conversely, over dehydrated tissue, due to prolonged alcohol step can be dry and hard to cut.

Clearing to remove the dehydrant

Toluene, xylene, and chloroform are the commonly used clearing agents, although xylene is less preferred due to its hazardous nature. Therefore, less toxic alternatives, such as isopropanol, have gained popularity. However, eliminating isopropanol during infiltration requires higher wax temperatures.

Infiltration with molten paraffin, to ensure successful tissue embedding

Paraffin based waxes are most commonly used and these are a mixture of paraffin wax and additives such as polyethylene or styrene. The wax used should be of good quality, as poor quality wax is unable to provide proper support to the tissue, resulting in blocks that are difficult to cut.Vacuum automated systems provide the advantage of rapid and efficient wax infiltration.

Improve sample preparation
  • Using appropriate processing schedule

Appropriate schedule should be chosen depending on the tissue type and size; for example,less time is required for small endoscopic biopsy and more for large, fatty specimen.Over processed endoscopic biopsy samples become brittle can develop fine cracks throughout that are visible under microscope, which only becomes worse with poor microtomy technique. Under processed large fatty specimens appear fragmented under microscope and lack nuclear definition. Furthermore, specimens should be properly fixed to avoid zonal fixation, that occurs when the sample gets formalin fixedfrom outside and alcohol fixed in deeper areas, resulting areas of intact and hemolyzed cells. Nuclear streaming is another frequently observed phenomena that occurs when water in the tissue moves out too quickly, resulting in “squeezing” the cell, thus leaving a final appearance of the nuclei in a “streaming” configuration.Thiscommonly observed problemcan occur due to incomplete dehydration, too rapid dehydration, or processing solutions that are overused and in need of changing out.

  • Maintain reagent quality

Tissue processing reagents should be strictly replaced as per the guidelines. Ignoring these, such as using diluted or contaminated reagents, can result in poor processing quality; for example, old formalin bottles in cold storage can be identified with the presence of white paraformaldehyde deposits. Specimens can show poor preservation when heavily contaminated reagents are used well “out-of-threshold.”Bouncing out of the tissue samples, especially uterus and prostate samples, of paraffin block during microtome sectioning or nonadherence to the slides or block can be seen due to residual water in the tissue as a result of improper dehydration and paraffin infiltration, and changing reagents and re-processing the tissue samples is recommended. Processing reagents saturated with water and/or paraffin saturated with xylene or isopropanol can also result in greasy looking tissue that separates or “explodes” when ribbon is placed in the water bath.

Take-home message

Fresh tissue samples are delicate and require processing in a series of reagents to enable proper sectioning. Each of these steps should be customized depending on the tissue type and reagents used. Reagent quality and processing schedule are key determinants of the outcome of staining.