Artefacts in tissue processing: A review

Artefacts can occur during dehydration due to

Improper gradient of dehydration: Diffusion current crosses cell membranes during fluid exchange if there is a high concentration gradient between the fluid inside and outside the tissue, increasing the risk of cell deformation. ^[6], ^[8]

Excessive dehydration causes the tissue to become hard, brittle, and shrunken, making cutting difficult and also interfering with the staining. ^[9]

Under or Incomplete dehydration results in inappropriate penetration of paraffin and block created is difficult to section therefore resulting in distorted or fragmented tissue sections which generates artefactural features.

Artefacts during clearing

Artefacts may form owing to over and under cleaning of tissue producing excessive hardness, and hence hinder paraffin impregnation of tissue making it harder to cut during sectioning. ^[6], ^[10]

Artefacts during impregnation

The purpose of wax impregnation is to rid the tissue of the clearing agent (wax solvent), allowing paraffin wax to fully penetrate it. The wax is then allowed to solidify to form a block from which sections are cut. Crystallization is the artifact that is formed during this process. Since thicker tissue absorbs more cleaning agent, it takes more molten wax to completely impregnate; therefore, repeated changes are needed. When a clearing agent contaminates melted wax, it results in insufficient impregnation, which during sectioning produces cracking and crystallization. ^[11]

Artefacts during embedding

During embedding processes, it is common to encounter artifacts resulting from poor orientation. These artefacts can cause damage to the microtome and tear the sections, making it difficult to study tissue under a microscope. ^[12]

Artefacts due to poor processing

Inadequate fixation may be the cause of a significant loss of architectural elements and clarity in loose connective tissue. These may result from mistakes made during the tissue processing cycle, such as using chemicals that are depleted, choosing the wrong reagents, or not switching solvents in a timely enough manner. ^[9], ^[10]

Sponge artefacts

These are seen in tissues placed in cassettes sandwiched between sponges. The tissue has angulated holes around its periphery, which are frequently triangular in shape and have smooth edges. Occasionally, a small intermediate channel bridging the separate faults connects paired angulated holes. The stromal matrix and tissue consistency are related to the extent of sponge penetration. For instance, sponge brushes tend to penetrate softer, loosely fixed specimens more than they do firmer, well-fixed tissue. ^[13], ^[14]

Loss of soluble substances

Fat solvents are used in the manufacture of paraffin wax, cellulose nitrate, and the majority of synthetic resin embedded sections. Consequently, as adipose tissue is processed, fat will separate from fat cells and manifest as void areas encircled by a cytoplasmic rim, such as lipomas and cholesterol clefts in odontogenic cysts. ^[15]

Orientation artefact

Improper orientation will lead to disorderly arranged histological features in slide. In order for all strata to be visible on the completed slide, the epithelial margins, subcutaneous tissue, and deeper layers must all be oriented such that they are flat to the bottom. To ensure that the sliced section presents a legitimate representation of the tissue submitted, all tissue pieces should be securely embedded into the bottom of the container when more than one specimen is being embedded. ^[12]