Histotechnology: A Blend of Art, Science, and Automation Denise Bland-Piontek, CTBS(AATB)HTL(ASCP) William Anim, HT(ASCP)

HISTOTECHNOLOGY Histotechnology is the detection of tissue abnormalities and the treatment of diseases resulting from these abnormalities.  A histotechnologist prepares human tissue for microscopic examination and diagnosis by a pathologist.  And persons specializing in this technique are known as Histotechnicians or Histotechnologists. https://www.youtube.com/watch?v=PYsN50h1Wbs https://www.youtube.com/watch?v=sBfA30Ouc8w#t=13

Surgical Specimen Accessioning The LIS Clinical Details / Requisition Accuracy Adequate specimen Proper Fixative 10% buffered Formalin fresh

Gross Examination: Description: LIS Specimen measurement Photo * future automation Cut section Scan Cassette

Frozen Sections: A procedure to perform rapid microscopic analysis of a specimen. Cryostat: microtome inside a freezer 20 minute TAT

Fixation: a process in which a tissue specimen is placed in a fluid that preserves the cells as nearly as is possible in their natural state. Properties of a Good Fixative: Provides reproducible and consistent results Stabilizes and hardens the tissue with minimal distortion of cells Fixatives kill the tissue by denaturation of the cell's chemical content What is denaturation? It is a change in the chemical, physical and biological properties of the proteins caused by alteration in structure As a result, the proteins usually coagulate, i.e., become insoluble In this manor, the molecules are stabilized

Figure : Solid Sertoli cell tumour Figure : Solid Sertoli cell tumour. Poor Fixation: Tissue preparation is the cornerstone of immunohistochemistry. One major issue that people agree on is that either poor fixation or over-fixation can be absolutely detrimental to a histological technique. Various fixatives also differ a lot in how they fix the tissue, and whether they are irreversible or not. The major fixative used in histology labs is 10% neutral buffered formalin.

Processing: “Tissue processing” describes the steps required to take animal or human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. After fixation tissue cassettes are placed on a tissue processor where by we replace aqueous formalin with alcohol in gradual sequence (70, 95, 100%) to make way for infiltration of paraffin wax. Processing removes the liquid and replaces it with a solid.

Clearing: Xylene Wax and ethanol are largely immiscible. We therefore have to use an intermediate solvent that is fully miscible with both ethanol and paraffin wax. This solvent will displace the ethanol in the tissue, then this in turn will be displaced by molten paraffin wax. Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to wax infiltration. A popular clearing agent is xylene and multiple changes are required to completely displace ethanol. Impregnating with paraffin.

Embedding: Now that the specimen is thoroughly infiltrated with wax it must be formed into a “block” which can be clamped into a microtome for section cutting. The specimen is very carefully orientated in an embedding mould fill with melted paraffin wax because the tissue placement will determine the “plane of section”. A cassette is placed on top of the mould, topped up with more wax and placed on a cold plate to solidify. When this is completed the block with its attached cassette can be removed from the mould and is ready for microtomy. It should be noted that, if tissue processing is properly carried out, the wax blocks containing the tissue specimens are very stable and represent an important source of archival material.

Microtomy: Creating great 5µ paraffin sections using a semi-automated microtome takes a great deal of skill and experience. A Histotechnologist feels the tissue density. Floating sections onto slides, the water bathe is tricky Flat, no air bubbles, no stretch or breaks, well hydrated tissue > high quality sections every time

Automated Staining: Routine stain > H&E The hematoxylin and eosin stain (H&E) is the most widely used stain in histology and histopathology laboratories. When it is properly performed it has the ability to demonstrate a wide range of normal and abnormal cell and tissue components and yet it is a relatively simple stain to carry out on paraffin or frozen sections. In histopathology a high proportion of cases can be diagnosed by an experienced pathologist using an H&E stain alone.

Coverslipping: To preserve and support a stained section for light microscopy, it is mounted on a clear glass slide, and covered with a thin glass coverslip.

Special Techniques:

Histotechnologist are also artists.

Our laboratory offers 25 standard special stains Special Stains: use a variety of dyes and techniques to stain particular tissues, structures or pathogens (such as bacteria, minerals, fat, or organisms) to assist pathologists with tissue-based diagnosis.   To detect the presence of acid-fast mycobacteria in tissue sections. To demonstrate acid mucopolysaccharides. To demonstrate sulfated muco-substances. To differentiate between neutral and acidic muco-substances. To demonstrate nerve fibers and the presence of neurofibrillary tangles and senile plaques in Alzheimer’s disease. To demonstrate the presence of amyloid The demonstration of spirochetes, Helicobacter pylori, or the causative organism of legionellosis. To detect the presence of Mycobacterium leprae in tissue sections. To demonstrate argentaffin substances. To permit identification of Helicobacter pylori in tissue sections. To permit differentiation of cells present in hematopoietic tissue. The demonstration of fungal organisms in tissue sections. The demonstration of Gram-negative and Gram-positive bacteria in tissue. To demonstrate glycoproteins, carboxylated and sulfated mucopolysaccharides. The detection of ferric iron in tissues. Staining of “epithelial” mucin in tissue sections The demonstration of neutrophils. The demonstration of neutral lipids in frozen tissue sections. Degenerating material containing fat, such as cell membranes or myelin, may coalesce into fat droplets that are demonstrable with fat stains, and tumors arising from fat cells can be differentiated from other types of tumors. The demonstration of polysaccharides, neutral muco-substances, and basement membranes. The demonstration of glycogen in tissue sections. The demonstration of reticular fibers. Differentiate between collagen and smooth muscle in tumors and to identify increases in collagen deposition. To identify the presence of calcium in tissue. AFB, PAS, Trichrome, Retic, Congo Red, Steiner, Fontana, Von Kossa, Elastic, Giemsa, GMS, Alcian Blue, Fite Gram…..

Immunohistochemistry (IHC) is the process used to visualize, identify/ classify tumour or viral cell types in tissue by targeting antigens with the application of antibodies. Marker Sec. Antibody Primary Antibody Tissue Antigen Our laboratory performs over 300 different markers, including some probes for chromogenic in situ hybridization. B cell Lymphoma – CD20

Quality Control Slide Review: Histotechnologists are expected to identify tissue structures under the microscope as well as understand the relationship of positive and negative internal control structures per test.

Where is the Full Automation Histopathology has had several advances in technology, but for the most part little has changed: https://www.youtube.com/watch?v=TLm37BbR1mo Vendors are finally taking it serious Full line automation expected over next decade

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