Extraneous Tissues in Surgical Pathology Practice

Introduction

“Floaters”, or extraneous tissues, or contaminants, are the part of the surgical pathology laboratory life. Are they preventable? Yes. Can they be completely eradicated? No. As an error in human endeavors they come with the territory of multiple predominately manual procedures in the laboratory. With the dramatic increase of amount of biopsies, floaters, including undetected, can influence and jeopardize the diagnostic process. They are the real problem in surgical pathology.

Despite surgical pathology practice is dealing with floaters on every day basis, there is no any systematic study of them focused at their prevention. In 1994, the College of American Pathologists (CAP) conducted Q-Probes study in 275 laboratories on extraneous tissues in surgical pathology which remains the solely statistic research on this subject. The results were published in Archives Pathology and Laboratory Medicine in 1996 (1). The CAP Q-Probes showed that there was an overall extraneous tissue rate of 0.6% of slides in the prospective study and 2.9% of slides in the retrospective study. In the article’s Comments, the authors wrote: “We know on no body of literature describing laboratory techniques that would lessen the possibility of tissue contamination.” This remains true now, more than ten years after.

The internal habits of different laboratories, while specific for each laboratory, have an element of similarity in adherence to basic standards. Each laboratory takes upon itself the implementation of them. The floaters topic periodically creates an explosion of discussions on the HistoNet, but the recommendations do not extend cleaning the waterbath and the embedding stations forceps wells.

The presented article suggests a systematic approach to floaters prevention in surgical pathology with attention to detail at every step of processing practice. One of the methods of Root Cause Analysis has been used.

Root Cause Analysis method of error prevention

A floater is an undisputable processing error. Root Cause Analysis (RCA) is one of the methods of preventing errors in medicine. The Eindhoven Model of Incident Causation, originally developed for chemical industry (2), has been applied to medicine in the end of 90th (3). A modification of the medical version of the Eindhoven Classification Model, as the table method of Root Cause Analysis, has been used for specimen misidentification prevention during accession and grossing in surgical pathology (4). Although the specimen misidentification is an unfortunate “event” rather than processing error as a floater is, the principles of Root Cause Analysis can be applied in floaters prevention.

The Eindhoven’s original taxonomy of Root Cause Analysis breaks up in three main causes of error: technical failure (equipment and software), organizational (policies, procedures, and protocols), and human operator (mistakes and violations). In practice, the distinction between technical and organizational causes, as well as human part in both, is blurred, although they are divided for categorization sake. All categories are overlapping each other as in Venn diagram.

Depending on the laboratory situation, the sequence of prioritizing and significance of categories in different practical applications can be changed. The method gives an opportunity for a systemic approach with constructive suggestions for incremental improvements. Floaters are minuscule by nature. Prevention methods also are not gigantic rather meticulous.

Systematic approach to floaters minimization

In opposite to original Eindhoven’s RCA model, the categories in floaters prevention follow a different sequence. The organizational part plays a dominant role. The technical details can only support, though significantly, the organizational efforts. And, of course, everything ends up with the concrete human participants.

Organizational

Floaters prevention is not a temporary campaign, but a permanent system of consistent organizational measures. The last thing should be a bureaucratic approach to satisfy some inspection requirements.

There are three main organizational components.

A/ Standard Operating Procedure (SOP). Although SOP cannot reflect every detail of processing, it should have materials which include floaters prevention. The update of these materials draws attention to details which are in the core of floaters prevention.B/ Occurrence Report. Independently of its diagnostic significance, an occurrence report of every floater incident should be the first organizational measure. There can not be any threshold for establishment of a protocol of floaters registration. Each of the occurrences can be sporadic, discovered or solved by different occasions, but if a protocol of follow up them were established, some practical conclusions might be made to prevent a pattern or a significant by consequences floater incidents. The documentation should be maintained by histology supervisor and evaluate by surgical pathology director.

C/ Protocol of investigation of the occurrence. Every floater occurrence ought to be investigated. The protocol of floater occurrence investigation is very delicate and often complicated keeping in mind the natural attitude for denial. The pattern of investigation can be delineated in following sequence: slide- block- embedding log- gross description- grossing log- requisition on the container- requisition chart. It means backwards as the specimen processing is done. Following the sequence of specimen’s grossing and embedding procedures according to logs is crucial in the investigative process. The

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management ought to insist on maintaining logs, both in grossing and embedding. Often embedding technicians do not follow the grossing log for many technical reasons that can be an impediment for the investigation. The surgical pathology management should involve minimal but most competent workers in the floater occurrence investigation otherwise finger pointing only obscures the picture. Supervisors should not delegate this responsibility to the committee or somebody else. Committees with their collective irresponsibility are not helpful, rather a pretension of actions.

D/ The periodical documentation follow up by histology supervisor and surgical pathology director.

Technical

If we exclude completely rare occasion of floating tissue debris in the tissue processors, there are only three areas of possible the floaters generation: grossing (sampling, cut-up), embedding, and microtomy. Let’s look at the technical category in these three areas separately, as the direction of specimen’s processing goes from grossing to microtomy. Each area has its technical particularities as far as floaters prevention is concerned. In floaters prevention, the technical category is more about tiny details of practice than hardware and software.

Grossing

Grossing is the main area where diagnostically significant floaters are generated. Its role in floaters is underestimated because the histology technicians are closer to the slide where the floater is revealed.

The technical part of preventing floaters while grossing is in providing appropriate conditions for good working practices. This is not about commonly accepted and understandable rules of ordinary cleaning instruments and keeping the working place neat. These obligatory common rules can prevent floater in large specimens which are diagnostically much less challenging than in biopsies. The latter require more detailed set of techniques.

The surgical pathology does numerous biopsy specimens by handling often almost identical material under the pressure of turn around time (TAT). Because the most significant in diagnostic sense floaters are generated while grossing biopsies, most preventive measures are concentrated on this type of specimens. The organization of a working place for biopsies is different from large specimens. This circumstance often slips away from the management attention.

Technical component:

a/ Processing board

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The most rational, as far as a prevention floater is concerned, is a cafeteria like plastic tray as processing board (Figure 1). The tray should have a cutting/working area (better with a glued rubber) and space for necessary instruments and gadgets (paper napkins, filtration paper, etc.).

Link to www.grossing-technology.com “Instruments& Gadgets” Cafeteria tray

The change of the tray’s set up (napkins, paper towels, pads) after every specimen is the ideal but it is unattainable, of course. However, soiled napkins or other accessories can not be tolerated.

b/ Filtration As one of the technical procedures in preventing floaters, filtration is an optimal method. Although the main purpose of filtration is assuring completeness of submission, as well as faster processing, minimizing any use of instruments can substantially contribute to floaters prevention. There different modes of filtration. Among them filtration through a special fabric with a diaper like background is the most effective in this author’s experience (5).

c/ GlovesTissues that are stuck to the gloves are potential source of floater contamination. While grossing, gloves are loosing their smoothens, become filthy and inevitable attract tissue which might be the source of floaters during cutting, wrapping, closing cassettes or other manipulations by unpredictable flicks of attention and other unfortunate circumstances. It sounds trivial, but washing and changing gloves is on of the technical part of floaters prevention. Latex or nitrele, pale beige color, not oversized gloves are optimal.

d/ Instruments and gadgetsBlades should be rinsed after of every specimen cut. What about change them? It is impossible, but if there were a sticky tissue (tonsil, large papillary urothelial or ovarian tumor, villous colon polyps), the change is mandatory. While changing, the special attention should be paid to the place of the blades insertion in the handle, which might contain some tissue material. Rinsing of the blade holder separately is useful.Forceps are the most possible culprits of a floater. Rinsing forceps after every specimen is obligatory. However, some tissue might remain in the forces serrated tips that may require the use of some gadgets. For example, a metallic brush under constant flow of water can be effective. There should be condition as permanently running water. One of the gadgets for forceps teeth cleaning is presented on figure 1 (6).

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Figure 1. Brushes for grossing forceps cleaning.

www. grossing-technology.com link to “ Instrument & Gadgets” Forceps Cleaning Cylindrical Brush

Disposable plastic pipettes for transfer some biopsies, like GI specimens, should be rinsed after every specimen. They become sticky requiring replacement many times during the day.

Some inks, especially Hematoxyline, which are used in grossing to make biopsies more visible for embedding, can be a medium for fungi contamination. Every day change of these inks is a part of proper working practices. Embedding

Embedding is always blamed for a floater that is a many occasions true. Solidifying paraffin makes small biopsies stuck to gloves, hands, and instruments during unwrapping or opening filtration bags. However, technical measures to solve floaters prevention are simpler than in grossing. The main of them is cleaning the instruments heated wells not only in the end of the working day that is obligatory, but while embedding session as well. Disposable cotton tips should be at hand.

Technical component

A/ One specimen at ones without any exception.B/ Regular cleaning with KimWip paper of the embedding station surface with attention to the grooves around the cooling plate.C/ Cleaning the instrument’s heated wells with Q-tips or plastic dropper to suck off all paraffin remnants.

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D/ Cleaning embedding instrument in microbiology loop incinerator. (Bunsen open fire is verboten) to burn the paraffin in their tips while embedding.

Embedding station, which in general haven’t been changed for decades, are not focused on floaters prevention, although some improvements could be made. For, example, to have the heated well for instruments of different diameters to accommodate different of types of embedding forceps to ensure complete and fast melting of the paraffin with a possible tissue stuck to the serrated branches. High thermal conductivity metallic removable inserts for the wells can be made for easy cleaning of them by a paraffin clearant reactive. A special heated high thermal conductivity metallic brush can be used for the embedding forceps cleaning. The brush allows the embedding person to clean the forceps after every embedded specimen. The warm bristles effectively clear the forceps’ teeth from the remnant paraffin. The melted paraffin goes down in the well which is cleaned in the end of the embedding batch (6).

www. grossing –technology.com link “Instruments & Gadgets” Cleaning Brush for Embedding Forceps

Microtomy

Microtomy traditionally is considered as the first culprit of floaters although it is not true.The CAP accreditation questioner specifically asks (ANP. 23350), if flotation baths are clean and well maintained and there is a procedure for prevention cross-contamination of paraffin sections in the bath with a note of “particular importance are periodic water changes or blotting of the water surface so that sections from patient block are not inadvertently carried over to another case so-called “floaters” or extraneous tissue.”

Among “technical” components are simple trivial actions:A/ blotting or, better skimming the water bath with a lint-free tissue, e.g. KimWip like paper. The principle is that the paper should not be glossy water destructive and too soft easily saturated by water; B/ every day cleaning the water bath;c/ periodical general water bath cleaning. Human

All technical categories are only valuable if the human part is included in the equation. Of course, every floater is a human error, definitely unintentional and embarrassing. However, it is not enough to decry about attention, accuracy, and meticulous diligence. Besides these obvious notions, the main contributing factor in floaters prevention is theprofessional education that provides understanding by workers details of methodology and the nature of the processed tissues. If the grossing person does not have definite knowledge of what he/she is doing, there will be slips in following written and unwritten rules of processing. Professional education allows the worker make inevitable deviation from strict following these rules without compromising quality of processing. And again,

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training of proper work practices, encouraging following the protocols and supervision of them is essential.

The Root Cause Analysis categories are summarized in the table 1. Every component is presented by short denotation.

Table 1. Route Cause Analysis summary of floaters prevention

category componentorganizational a/ floaters prevention in SOP;

b/ occurrence report;c/ investigation protocol;D/ follow up documentation.

technical Grossinga/ tray as processing board;b/ filtration;c/ gloves;d/ instruments and gadgets.

Embeddinga/ cleaning of the station (KimWip) and molds (clearant)b/ cleaning the warmed wells from paraffin debrisc/ cleaning instruments (incineration, brushes).

Microtomya/ blotting or skimming the water bath (KimWip)b/ every day cleaning of the water bathc/ periodical general cleaning of the water bath.

human a/ professional education;b/ proper work practices training.

Discussion

Can an extraneous tissue be placed outside the laboratory? As a rare occasion such contamination can occur during the specimen, namely, the biopsy harvest when multiple biopsies are taken during a procedure and one of the biopsy fragments for some technical reasons was placed in an inappropriate transport container. But surgical pathology floaters are different from specimen mix-up or misidentification; this is a laboratory error of processing.

The term 'floater' came in surgical pathology laboratory from the step in histology processing when tissue sections are floated on the surface of a water bath before being mounted on a slide. A small fragment of tissue can break free from the paraffin section and float in the water bath until it is picked up with a section from another case.

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Although dictionaries give many meanings of the word floater, most of them are connected with something floating in the water (for example, the old British – a corpse in the river). Floater has mostly derogative meaning, a drifter. This linguistic excursion is indirect evidence that in common pathology laboratory perception the floaters are caused by failed cutting the slide. Histotechs in general accept this blame on themselves but in reality this is not the case. The floater generated during microtomy, namely in the water bath, is almost always on the periphery of the slide, easily detectable and removable. Although if a tissue ribbon is laid on the waterbath directly over the floater, the floater could be trapped beneath or immediately adjacent to the ribboned tissue. The heating after cutting stops the floater at this place. The recent study of microtomy water baths and stainers found from 3 to hundreds cells as cross-contamination in 8% of blank slides (7). These floaters are the most innocent in their diagnostic significance. They are different from other groups of floaters, which are not floaters at all by their origin. The definition of extraneous tissue is more precise, but for common use ‘floater’ is more acceptable, although it deflects attention from the main sources of contamination as grossing and embedding.

In a simplistic “classification”, all floaters can be divided in four categories. This is not for taxonomy, but rather to distinguish them by actions after the floater is detected.Nuisance. For example, a fragment of a liver tissue is attached to the gastric biopsy slides periphery or even in the middle of the slide and incorporated in the block. The pathologist can ignore it, but usually with some grumbling the intruder can be taken out from the slide or the block is melted following by a similar procedure. The end of the problem.Manageable problem. The floater is definitely distinguishable from the rest of the slide, but requires additional clinical and pathology consideration.Serious problem. It requires identification phenotyping if there were a suspicion of a contamination like in knife –induced focus of vascular invasion. The main thing is to suspect a floater. The problem always can be solved by using FISH and molecular genetic techniques additionally to general morphological picture (8, 9, 10, 11, 12, 13).

An undiscovered floater can occur. Under conditions of mass biopsies production in modern surgical pathology a floater might be even not suspected. For example, in children’s gastroenterology with very similar biopsy material, when celiac disease is in question, this floater can be crucial for diagnosis. Although between the similar specimens a contamination occurs rarely, it is the most difficult to detect. The consequences of undetected “floater” can be serious. For example, a carryover from a lung tumor to a bronch biopsy which upon thoracotomy turned out to be histology only. ‘Vanished’ prostate cancer in radical prostatectomy specimen, when prostate cancer cannot be found after histology examination of the entire removed gland, might be also a result of a contaminated prostate needle biopsy (14).

There is a disputable area of a grossing contamination when a postoperative pseudo micro invasion of the cancerous uterus might be generated by mechanically transporting tumor into vascular spaces during the grossing process (15). In any event in this instance, an additional attention to cleaning of instruments during grossing would be helpful to dissolve any clinician’s concerns.

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There is a rational consideration behind the attitude to get rid of the floater on the slide by any cost. Although the floater would not have anything to do with the diagnosis and haven’t impaired the diagnostic process, the slide might go for a review. In a legal case, nobody can predict the outcomes. The presence of an extraneous tissue can cast a shadow on the diagnosis because the principle res ipse loquitur, the thing speaks for itself, works in this situation in its direct meaning.It seems that as it is right to eliminate the floater from the slide and the block as much as possible. It is definitely right never to mention the floater in the pathology report although the internal investigation should be always documented.

Actually, most floaters by themselves are more nuance than problem. However, their presence might be an indication of violations of proper working practices in the laboratory. This is the reason that careful documentation of them is not a bureaucratic endeavor. The common knowledge states that revelation of a small problem prevents it turning into substantial.

What area is the most responsible for floaters? This is not an academic question because it is connected with what to do for their prevention. Unfortunately, often it is impossible to decide either embedding is responsible for a floater or grossing. The mentioned already 1994 CAP Q-Probes on extraneous tissue concluded that in 92, 3% the origin of extraneous tissue was in the surgical pathology laboratory (1). However, grossing, embedding, and microtomy, all belong to surgical pathology. Although in the retrospective study 15.9 % of extraneous tissue were on block only, there is not clear where the extraneous tissue was generated. Real floaters which stem from the waterbath are rare. Most extraneous tissues are contaminants.

The Cap Q-probes article (1) included a table with a list of 15 possible sources of extraneous tissue in following sequence (gloves, dissecting surface, instruments, strainer, saws, scales, container, microtome, tissue cassettes, ink, fixative solution, specimen container, staining solutions, water bath, and towels). The problem is how to weigh their role in floaters contamination with possible practical recommendation.

There are proprietary protocols and methods of floaters prevention in different laboratories. The main point is in a systematic approach. The advantage of original Eindhoven’s RCA method is that it concentrates on constructive proposals of solutions depending on current circumstances in laboratory. For example, the bar-coding of blocks and slide labels becomes more and more popular while bar- coding of requisition forms is not a novelty at all (16,17). Although these technical improvements aimed on prevention of specimen’s misidentification, they can be used also for tracking floaters. They can be included in floaters prevention protocols by increments due to their cost. Three dimensional RCA model provides a template for permanent analysis and enforcement of protocols. Instead of generalities slogans, this method provides a “shopping list” of mandatory items as well as a “wish list” of additional improvements. Some of the components are trivial, but obligatory. Other are just optional but desirable. Together they lay down a foundation for floaters prevention.

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References

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