Vol. 19 • Issue 4 • Page 42
In the continuous flow of specimens associated with the histopathology lab environment, there has been increasing growth in small biopsy-related specimens submitted. Whereas two decades ago the overwhelming majority of specimens submitted in surgical pathology were larger hunk-like tissues often filling the inner cavity of routine processing cassettes, today we see the numbers of minute tissues overtaking the former specimen sizes.
Almost a decade ago Dr. James McCormick, pathologist and premiere innovator in histology equipment design, began conducting a five-year study of surgical specimen submissions. He noted that in 2001 the total submission of small specimens in the U.S. clinical market was approximately 40% of the total number of larger, hunk-like specimens. But progressively each year the margin of difference shrank as the larger specimen totals decreased and the small biopsy-related specimens increased. He further noted that these totals leveled off to being equal numbers in 2004, and that by 2005 there were more biopsy specimens submitted than large tissues (Fig. 1).
New Challenges
Greater complexities associated with small tissues have challenged laboratories to not only modify and/or develop new procedures, but also implement some form of risk management program to avoid mistakes and loss of tissue. This creates a need for a greater understanding of the science of small specimen management-one that encompasses all areas of handling such as specimen procurement, transport, fixation and processing, microtomy and staining.
Additionally, with the enhanced skill and focus needed in the area of microtomy of small tissues, this also creates a need for cross-training to accommodate the increased workload. Historically, sectioning biopsies has been an assignment reserved for the most skilled microtomists, but in today’s workflow, managers do not have the luxury of only using a select few technologists in this area and must have multiple personnel competent enough to handle small specimens.
The change in specimen submission from hunks to snippets has been driven by need. With the formerly common utensils used for tissue procurement being scalpel and forceps, medical technology has developed more precise, less invasive means of isolating and extracting localized areas of pathology in tissue. Physicians now have the ability to pinpoint an area of interest in the body, then take a small snippet of tissue only. Utensils are specialized for precise procurement of specimen through procedures such as needle and punch biopsies, fine needle aspirations, etc. Less hospitalized patient care is needed due to the localized procedures, so outpatient visits have replaced the need for many overnight stays, resulting in quicker diagnoses and a more rapid turnover of patient appointments. We have systematically seen an increase in patient cases that also has affected the rise in biopsy submissions.
Fixation & Labeling
Once a specimen has been procured, tissues must be placed in some form of tissue ðfixative as soon as possible to prevent autolysis. When there has been a cessation of blood flow to cells, as in tissue biopsy extraction, progressively the cells will produce enzymes to break down and autolyze the tissue. This process is much more rapid in small specimens, particularly thin, delicate, tissue structures such as the alveoli sacs of the lung and mucinous tissues such as in the gastrointestinal tract. Laboratories must have a system in place where the histology lab can review, monitor and document that appropriate collection procedures are being employed, even in off-site situations such as physician office laboratories. Supervisory and management personnel in the laboratory can oftentimes assist in managing the risk of improper handling by ðdeveloping documents to be filled out at the specimen collection site. These documents, along with proper staff orientation, would assure that correct labeling and transport procedures are followed.
Proper labeling of specimen is essential in histology as a rule; however, small tissue biopsies present an even greater challenge because in many cases individual focal poðints from the same organ must be differentiated and submitted independent of one another to give the pathologist information on specific areas of disease. A long-standing practice in gross pathology has been to separate like biopsy specimens cases from one another by logging in and inserting a larger specimen case in between the two biopsy cases. A number of ðhigh-volume laboratories, though, are 100% biopsy related, dealing only with tissues such as prostate needle biopsies, gastrointestinal biopsies and other small specimens. In these cases, sample integrity is maintained by thorough documentation, a reliable electronic tracking system and constant monitoring of the specimen flow from start to finish. Enhanced methods of specimen tracking have been implemented in the field, such as barcoding to immediately access patient data, and there is new research and development in special cassettes that have an electronic chip tracking device inserted with data specific to that patient specimen.
Communication
In reviewing standard quality assurance procedures in most histopathology laboratories, one of the primary areas of risk is in ðcommunication. Managers must assess the risk level at each stage from specimen collection to send out. Fig. 2 demonstrates a common pathway for surgical specimens, which, if proper details are not communicated from one stage to another, can easily result in information loss. The increased frequency of remote collection sites and the use of courier systems elevate the need for precise communication.
Cassette Design
The evolution of specimen containment devices has progressed to help reduce and/or eliminate the risk of tissue loss during specimen processing; recent design has seen a focus on cassettes specifically designed for small biopsy specimen. The new designs had to first accomplish the issue of security and ensure that even minute specimens would not escape from the compartment. Special “action seal” construction has been developed to lock in specimens and not open until manually induced (Fig. 3).
Additionally, a compartment that not only contains the specimen but allows for sufficient fluid exchange during processing reagent cycles emerged. One design, for example, incorporates a metal screen or mesh-like material. The efficiency of this design must be used in context with an understanding that the surface tension created by the minute pore size of the mesh oftentimes causes air bubbles to be trapped inside the cassette during processing. This can in certain circumstances compromise processing efficiency. To circumvent this, the user should use some form of mild agitation of the cassette when first introduced into solution to break the surface tension and help evacuate air bubbles.
Another model biopsy cassette was designed with a basket compartment interior with sloped walls to reduce surface tension and make tissues more accessible in the corner of the cassette. The ðmillipore holes are designed as truncated pores with a slight ‘V’ shape. The hole size at the bottom of the cassette is slightly smaller than the interior of the cassette. The hydro-dynamics of this design pulls fluid in and forces air out. This model eliminates the need for sponges, biopsy bags and other interior containment devices (Fig. 4).
Tissue Processing
In tissue processing, biopsy specimens are more susceptible to over-processing from excessive dehydration, prolonged exposure to xylene and heat artifact. This necessitates special biopsy runs optimized for small tissues. Excessive dehydration can create one or more issues such as total removal of bound water, fragmentation of molecular bond and disintegration of the protein molecule. Any of these will result in brittle fragmentation and flakes during microtomy.
With the advent of reference labs doing 100% biopsy tissue, additional reagents and methods have been employed to achieve rapid processing and turnaround time. Alcoholic fixatives are often used with biopsies to initiate the dehydration process while tissues are fixing. Heat inducement (microwave or other xylene-free applications) is also designed to speed up processing. These must be closely monitored, as alcohol and heat both act as catalysts to the dehydration process and can quickly render the tissue to a hard, crumb-like fragment.
Another caution to observe is the combined use of blended alcohol products with alcohol recyclers. While alcohol recyclers perform superbly with ethanol and reagent alcohols, commercially blended alcohols traditionally have a higher mixture of methanol and isopropyl. Approximate percentages of commercially blended alcohols are reported around 44% methanol, 16% ethyl alcohol and 40% isopropyl. In the alcohol recycler, each boils off at different ðtemperatures and subtly, each time the reagent is recycled, will yield a slightly higher percentage of methanol and less ethyl and isopropyl. Progressively this can render a solution less effective as a dehydrant, resulting in varying tissue profile, indistinguishable nuclei and loss of nuclear/cytoplasmic contrast in staining (Fig. 5). Monitor the number of times your lab recycles these types of products so as not to practice indefinite use.
Embedding & Microtomy
Embedding and microtomy carry the same sense of importance as in larger sized specimens except that there must be a heightened awareness to transfer and properly orient all pieces from the cassette to the embedding mold and to practice great care during microtomy so that tissues are not cut away during face-off or sectioning. Heated forceps help facilitate the embedding process, but they should have a fine point design to enhance dexterity. During embedding, if the newer
design, small millipore biopsy cassettes are used, air pockets can form in the cassette. This is due to the millipores cooling down and impeding paraffin from filtering through into the cassette on the final pour of embedding medium. Make sure the cassette stays hot on the hot plate of the embedding center until just before filling, then fill the final pour with the cassette very close to the paraffin dispensing nozzle. The force of the paraffin this close to the nozzle will help diffuse the medium through the pores and into the cassette mold.
Staining
The final area of risk to be discussed is routine staining, primarily that of the hematoxylin and eosin (H&E) stain. With most H&E staining in surgical pathology, a well-optimized staining protocol is usually sufficient for all of your tissue specimens. However, because of the less dense, minute nature of small tissues, this protocol can frequently cause over-staining of biopsies. The nuclear and/or cytoplasmic staining may be too intense, requiring a reduction in incubation time for the H&E. Some specialized areas of pathology (such as dermatopathology) many times have a preference for a darker, heavier staining effect in nuclear detail. In these cases either a longer staining time in hematoxylin or the use of a stronger hematoxylin is generally a subjective preference. In any event, care must be taken to adopt a protocol that is best suited for the tissues being used as well as the pathologist reviewing the slides.
With a greater demand for diagnoses of minute tissues instead of bulk specimen, there must be a response in the industry to develop methods, procedures, instrumentation and consumables designed to optimize the diagnostic capabilities of biopsy tissue.
Skip Brown is manager, Clinical Applications & Training, Leica Microsystems-Biosystems Division.
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