|Basic Anatomy and Tissue Identification|
The embedding histologist must practice to develop rapid visual recognition of tissue types and be able to determine the correct orientation for each specimen using their knowledge of anatomy and tissue sampling techniques. It is often helpful to consider the reason(s) that each specimen may have been selected for submission to histology. Also, remember that clear goals are outlined for each specimen to arrive at a diagnosis based on the type of specimen and the suspected reason for the surgical procedure performed.Gross dissection of tissue samples is determined by a pathologist or pathologist's assistant (PA). Many laboratories follow established protocols for the gross dissection of particular types and categories of specimens. Specimens are identified using a numbering system that associates a unique number with a specific patient, medical record number, and tissue sample upon receipt. The cassette containing the specimen from that unique patient and procedure is also identified with this number. This unique number is transcribed on the slanted top edge of the tissue cassette by either hand or machine, shown in the lower image. The slides prepared from the block in the histology lab from this cassette are likewise identified with this unique number, as shown in the image on the right.Tissue is selected for microscopic examination to document important details about each specimen. Some examples of specimens that may be selected for submission to histology include:All lesions grossly identified - Tissue between lesions may be included with the lesion to determine if the lesion is separate or interconnected to surrounding normal tissue.Representative sections of all normal tissues or structures that are not included with suspected disease-containing sections.All surgical margins, when appropriate.Frozen-section remnants for permanent sections.
|Basic Tissue Sampling and Use of Tissue Marking Inks|
Marking the tissue edges aids in the identification and correct orientation of the tissue pieces during embedding. It also assists in the correct placement of the intended surface toward the face of the block, which is the first surface to meet the microtome blade.Tissue markers must:Be insoluble in fixative solutions, processing solutions, and the embedding mediaRemain on the surface to be marked and not penetrate too far into the tissue interiorNot react with histologic stains or be over applied to obscure cells or tissue layersBe notably visible both macroscopically and microscopically Some examples include India ink, silver nitrate, and various artist pigments. Different tissue marking inks may be applied to mark anatomic or surgical landmarks. This will help orientation both during gross dissection, embedding, and in the final histologic section. The ink may be applied to color the entire outside perimeter of the specimen, or may be used selectively, using single or multiple colored inks.The application color and location will typically be dictated into the gross description.It is recommended to try to avoid red ink, since it may be difficult to see. Black or bright blue ink is often preferred. Ink marking examples include:Tissue TypeExampleSmall lesions present in a larger section of tissueThe lesion may only be visible on one "face" or plane of the section. In this case, one side of the tissue can be inked and specific instructions should be provided such as "embed with inked tissue surface up."Note that most histologists will want to relate any directional instructions to the block face, which is the surface that will meet the microtome blade first.Tubular structuresIt is sometimes helpful to submit the entire tube (vas deferens, temporal arteries, fallopian tubes) in the tissue cassette, with instructions to cut cross-sections before embedding. The lumen end can alternatively be dipped in tissue ink, to more clearly mark the opening for very tiny tubes. The lumen MUST be visible in the final section. Multiple fragmentsThe fragments may be inked on one edge, but should be embedded so that all pieces are kept in the same plane or level in the block so that one complete, representative section can be obtained.
|Spatial Reasoning and Visualization|
Histology is literally, "the study of tissue." Tissue sections cannot be produced which are completely accurate and representative "snap shots" of the tissue in its' living state. But instead, histologists seek to preserve the tissue in the most life-like state possible. In reality, what we are able to achieve is a somewhat distorted, yet permanent, representation of the tissue's living condition.Histologists must learn to be able to visualize how specimen handling, including gross dissection and tissue processing, affect the appearance of tissues at both the macroscopic and microscopic levels.A clear understanding of anatomy and the ability to spatially visualize the specimen in three dimensions is extremely helpful. The histologist must use this background knowledge and understanding to visual identify any alterations in the tissue appearance caused during gross dissection and/or tissue processing; Apply this insight for correct specimen orientation during embedding.In visually assessing and identifying tissue samples, note that: Some sections cut through tubes or invaginations are not large enough to show the entire lumen. Many cells and tissues are organized into partitions, which divide the structure into numerous smaller areas which may be transected in any given specimen.Many submitted specimens may not show all the structural details that are depicted in images in histology atlases or anatomy textbooks. You may become quite skillful at visually identifying many specimen types, but you MUST still verify the specimen type using an electronic or paper-based worksheet.
|Tissue marking inks must be clearly visible both macroscopically and microscopically.||View Page|
|Which of the following is NOT a purpose of the embedding process in routine histology? ||View Page|
|Specimens Containing a Lumen|
Any specimen that is a transverse section of a hollow structure or a specimen containing a lumen will almost always be placed in the block face to show the lumen or other opening in cross-section on the final slide. For any organs with very large lumen's in which the tube has been transected, you will embed on the cut surface to demonstrate the lining and layers present. Of special concern are specimens with very tiny lumen openings, such as temporal arteries or vas deferens. Since the lumen opening is so tiny, it is sometimes difficult to see. Some laboratories may lightly dip the end of the tiny tube opening in black ink. This indicates the "end" that should be placed down in the block face. Some laboratories may submit such tiny tubes whole and ask that they be cut into cross-section at embedding. This allows the histologist to clearly see the opening during dissection. Other means to flag these specimens can be by using an abbreviation on the cassette such as "vas" for vas deferens, alerting the histologist that the specimen contained will need to be embedded on end to show the lumen in cross-section.
|Gynecological (GYN) Specimens|
Several types of GYN specimens are routinely submitted to clinical histology laboratories such as cervical loop electro-cautery excision (LEEP) procedures, cervical cone, endometrial curettage, and vulvar biopsies. Although for most GYN specimens you will follow the same general guidelines as for other similarly shaped and sized tissue specimens, it is especially helpful to understand a little about the surgical procedures used to obtain these specimens and also the common methods for dissection used.Cone biopsiesThis procedure is a conical excision of the cervical canal using a laser or cold blade.The wider part of the cone is the ectocervix, and the tapered tip contains the endocervical margin.The ectocervix by convention is described as a clock face, with the most superior midpoint of the anterior lip designated at 12 o'clock.Sections will usually be submitted sequentially and designated in the gross description by their clock face orientation.These sections should be embedded so that perpendicular sections will be shown of the cut surface.Cervical LEEPThese related procedures remove less tissue than a cone biopsy and are obtained by electro-cautery of the cervical transformation zone.The specimen may be divided either perpendicularly or by using a radial dissection method.Endometrial curettageCurettage is a scraping method, in this case of the endometrial lining.The specimen obtained will consists of bloody fragments.These may be submitted in biopsy bags to contain the fragments during tissue processing.The surface of the bags or paper should be scraped lightly to remove as much material as possible.Embed to keep the fragments centralized in the block face and to arrange for the greatest surface area to be shown in one plane.Wipe forceps and all surfaces well after these specimens; it is easy to transfer the loose bloody fragments to other specimens.Embed all fragments with respect to the ink present with inked edges facing all in the same direction.
There are a few types of tissue specimens that may be encountered while embedding that will often be less than optimal, despite being processed in an adequate tissue processing program. These specimens include: Fatty tissue: It does not fix well or dehydrate easily and may be "wet" after processing. This will make a white appearance in many specimens when the block is formed due to the water present in the tissue which will not allow the paraffin to infiltrate. Unfixed or poorly dehydrated specimens will often shrink inward from the block face. This tissue may require reprocessing steps with additional fixation, dehydration, and/or wax infiltration.Large tissue: This includes pieces of any tissue type that has been submitted in sizes that are too large for the cassette. The tissue will often not process well, and deep grooves may be pressed into the tissue from the cassette that will have to be sectioned past to obtain a complete section.Specimens with inadequate decalcification: It may be possible to decalcify with decal solutions at your microtome station or use prolonged soaking to help obtain a section.Hair: It simply does not section well. It is best to try to remove hair if possible during gross dissection.Hard foreign materials such as sutures and staples: Items such as this will not section and will tear the tissue section during microtomy. It is best to remove these during gross dissection, but it may be possible to remove during embedding and placed on top of the cassette before it hardens.Sebaceous cyst contents: Quite often, this material does not process or section well. The material can sometimes be removed from inside the cyst and then placed on top of the block so it will not interfere with obtaining a good quality section of the cyst wall. Many of these issues are due to poorly dissected or handled specimens that are then submitted to histology in this less than optimal condition. In other cases, the tissue type itself may present some processing challenges and it may be difficult to optimize processing programs to be optimal for the varied tissue types that most histology laboratories receive. In all instances, you will be attempting to make adjustments and corrections to the best of your abilities for these shortcomings, so that the best possible microscopic section is able to be obtained.
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