Processing pathway of a surgical specimen

QUESTION: Describe processing pathway of a surgical specimen In histology a surgical specimen is any tissue that is brought to the histology laboratory prior to surgical removal of the tissue from a surgical patient by a surgeon and it could a biopsy or autopsy. Tissue processing in histopathological laboratory is a number of sequential steps that proceed prior to microscopy examination of the tissue and this process occurs between the arrival of the specimen to the laboratory and microscopy hence tissue processing is a secondary procedure in the histology process. All tissues must be adequately supported before they can be sectioned for microscopical examination except processing in frozen sections and is enhanced by a series of processes that changes the tissue structure hence promoting adequate rigidity that allow smooth tissue sectioning and not damaging the sectioning blade. Tissue processing can manual or automatic. The whole process of tissue processing is based on a principle in which there is diffusion of various substances into and out of stabilize porous tissues and the diffusion process results from the thermodynamic tendency of processing reagents to equalize concentrations inside and outside blocks of tissue, according to the Fick’s Law. These above principle explains how the rate of processing is affected by agitation, heat and vacuum. In histopathological laboratory tissue processing is sequentially based on complete fixation, dehydration, clearing, infiltration and embedding.

Fixation is the first process that precedes before everything and it a process that is done to preserve the surgical specimen from the process of putrefication and autolysis induced by enzymes. This process is important and it stabilizes the tissue by reacting with proteins and it also enhances penetration of the stains to be used and the major achievement is adequate rigidity of our surgical specimen. Different fixatives are used and they encompasses of formalin, acetic acid, osmium tetroxide, picric acid, mercuric chloride, ethyl alcohol, potassium dichromate and glutaldehyde as simple fixatives. The most common used fixative is 10% neutral buffered formalin and the volume of our fixative should be 15-20 times more to our surgical specimen and if is the specimen arrives to the laboratory with inadequate fixative it should be reported and addition of fixative is done to ensure adequate fixation. Adequate fixation with formalin occurs for approximately 8 hours for large tissues. It is important not to overfill in tissue cassettes to ensure fixation and very thick tissues need to be sectioned to ensure penetration of the fixative. 30 minutes is adequate provided our surgical specimen is a biopsy.Incompletely fixed tissues after dissection should stay in the first container to complete the fixation. Therefore, improper fixation will definitely affects that precedes up to slide preparation, hence fixation should be checked if it is properly done. After this process the tissue is placed in a tissue cassette. Labelling of tissue cassette is done prior to tissue being placed in a tissue cassette.

In some tissue processing steps, the processes can be automated example in dehydration, clearing and infiltration occurs automatically in a tissue processes overnight for 16-18 hours.

Dehydration is proceeds immediately and it is gradual removal of water from our surgical specimen. Water must be completely removed to allow the process of infiltration to occur because the wax is immiscible with water hence complete water removal. The most common dehydrating agents are ethanol, isopropanol, methanol and acetone. However, ethanol and isopropyl alcohols are used. The actual processes occurs by submerging the tissue for 3 minutes each in 70%v/v, 2×95%v/v, 3×99% then absolute alcohol respectively. This processes lessens the shrinkage, hardening and distortion of tissues brought about by diffusion currents. During this procedure various cellular components are dissolved by dehydrating fluids. For example, certain lipids are extracted by anhydrous alcohols, and water soluble proteins are dissolved in the lower aqueous alcohols hence dehydration also important.

Clearing follows immediately after dehydration and this the removal of the dehydrant with a substance that is miscible with both the dehydrant and the clearing agent. The tissue becomes translucent hence the term clearing. This process improves the refractive index of the surgical tissue. Clearing agents commonly used in histology are xylene, chloroform, toluene and benzene but the most commonly used is xylene. This process must be well monitored and prolonged clearing can lead to tissue brittleness and hardness that might lead to difficulties in tissue sectioning and inadequate clearing leads to improper penetration of paraffin wax and might reduce the translucency of the tissue. Clearing in xylene is done in 3 jars of xylene by complete suspension of the tissue in the cassette for 3 minutes in each jar.

Immediately, infiltration takes place and it is a process whereby there is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally embedded and this process is also called interpenetration or impregnation. Paraffin wax is commonly used and there are common additives like beeswax, rubber and plastics. During the process, tissue should be removed from the molten wax, as soon as possible, since the heat required to keep the wax molten (56- 60 °C), will cause hardening and shrinkage. Wax temperatures must be monitored, since excessive heat will damage tissues. Thus, this process must not be too prolonged or too short hence must be monitored.

Embedding is a process which involves the encasing of the tissue in the infiltration media, and letting the wax solidify and this inter-dependent to infiltration. It is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning. In the embedding process, the proper orientation of the specimen is essential and examples of orientation are flat, on-edge and on-edge. Instrument used in embedding have a heated reservoir for molten wax storage, heated stage where tissues are oriented in the mold and a cooling stage where tissues are rapidly cooled to ensure crystal size and then moulding proceeds.

Microtomy is then done to section the processed surgical tissue in which the procedure is semi-automated. After thin sections have been made which are 4-6 µm thick they are attached to slides then heat fixed to allow adherence of the tissue to the slide. Bringing sections to water by gradual rehydration of water to tissues by subsequent suspension of slides for 3 minute intervals in 3 × absolute alcohol, 2×95% alcohol and then 70% alcohol. Staining then occurs after the whole process then mounting with D.P.X occurs and the final product is a slide. REFERENCES

Histology: An introduction to tissue processing Lecture #14.
Bancroft’s Theory and Practice of Histological Techniques 7th edition 2013 Edited by S. Kim Survanna, Christopher Layton, John D. Bancroft Churchill Livingstone Elsevier.