The Accession Triage

Specimen receiving unit with the accession person as a main player is often underestimated one of the central parts of the grossing room in the surgical pathology division. Smooth work flow with concentration on accuracy and quality of processing is the main indicator of efficiency of specimen accession. This is a place where the many severe troubles are generated in surgical pathology grossing.

The accession work in surgical pathology requires predominately common sense and appropriate medical and pathology education. Common sense is not teachable, but medical and pathology knowledge can be acquired. These remarks are intended to make a blend of both medical and pathology training for initial treatment of diagnostic material upon receiving at the pathology department.

Different institutions understandably have different work load, specialization, established traditions, and simply a concrete cast of players. Nevertheless, there are common principles that ought to be followed otherwise the quality and speed of processing suffers or the specimen can be even ruined.

Perhaps, it would be superfluous to mention that the specimen must be checked for labeling, consistency of the requisition form and specimens, if it were not for so many occasions when these inconsistencies should be clarified. For many practical reasons these discrepancies should be straighten out as soon as possible, because delays eliminate the immediate participants on the clinical part of the specimen that complicates the situation. Sometimes, the specimen requires immediate processing for many reasons before the discrepancies are straighten out that request from the accession personal adequate training and common sense to send for processing with a provisional accession.

In essence during the accession the specimen acquires a surgical number but the work includes much more elements. Big laboratories replaces community hospitals laboratories like malls mom and pop shops. The specimens often arrive in droves. These circumstances require established protocols of specimen’s handling in different situations.

The main aim of the accession as a medical action is triage in literal meaning of this word. as setting priorities. The quality of the accession unit and the accession person is The first step reflected by how triage is executed. The accession triage determines the direction of examination and grossing triage. The accession triage should be under supervision and direction of grossing person, in institution with residency under supervision the resident in charge.

The accession person has to solve following immediate triage questions:

Does the specimen belong to surgical pathology?

Not so rarely microbiology samples are sent to surgical pathology as a division of pathology, or together with surgical pathology specimens, or, the worse case that surgical pathology is required to take material from the main specimen and send to microbiology that is wrong by essence. The accession person should alert the grossing person about the letter situation to solve the problem keeping in mind the sterility of collecting the material for microbiology.

Of course, the cytology material often is sent to surgical pathology by mistake or intentionally, especially in a case of PAP that is distinguishable by the specific container’s design. Sometimes, the material sent to surgical pathology is apparently insufficient, that requires attention of the grossing person as soon as possible to direct the examination to cytology or alert the clinicians without an unnecessary delay.

Cytogenetics, molecular biology specimen often get lost at surgical pathology specimen receiving that require proper recognition of them with immediate redirection.

What kind of speed of processing does the specimen require?

We do not discuss the rush  “irrational exuberance” epidemic that occur in some institutions or departments. There are three main definitions of the expedition of the specimen processing in surgical pathology: Frozen section (FS), Rush (same day or overnight), and Routine.

Frozen section priority is understandable. The work of the accession person in this situation should be under strict control of the pathologist, resident in charge, or grossing person. Accurate timing (better by computer) and following the sequence of the specimens is essential if they arrive as multiple in a short time, or from different patients.

Routine processing now becomes more blurred and unclear due to the lamming rush of the pathology departments to same day processing of biopsies. The discussion of this big issue is out of the scope of this review, but this is the every day reality of processing in large laboratories. The accession has to deal with prioritizing some specimens out of the routine group for routine/rush processing.

The real triage problem is a “Rush” specimen. No all “Rush” are meant equally, some are real “Rush”, some are “Rush”, but can be postponed, some are phony “Rushes” when the clinician meant “not too slow”. There are clinicians who are abusers of this definition thinking that their specimens have a special priority value, as well as situation when the mark “Rush” in the requisition form is directed to the delivery personal for immediate specimen’s transfer to the pathology department. Needless to say, that some “Rush” specimens are imposed by internal pathology department considerations, including evening stay, weekend, children’s soccer lesson, and etc.

Real “Rush” are specimens for transplant acute rejection, in opposite to annual surveillance for transplant rejection that is not a “Rush” specimen although often is considered as a “Rush”. Another example of a real “Rush” is acute necrotizing skin

( Toxic Epidermal Necrolysis TEN)   that has its protocol that includes “Rush” examination.

A renal biopsy for acute renal failure is undisputable “Rush” same day specimen.

Bone marrow biopsies usually arrive in fixative that requires monitoring of the fixation.

What kind of preliminary treatment does the specimen need before processing?

Although preliminary treatment is the prerogative of the grossing person, it starts during accession triage. This is the question of preservation the specimen. It is discussed in detail in the post “Introduction to Biopsy Grossing Techniques.”http://grossing-technology.com/newsite/home/grossing-techniques/introduction-in-biopsy-grossing-techniques/

This article does not touch such crucial question as mixed  up specimens. This question is discussed in the article “Root Cause Analysis in Specimen Misidentification Prevention in Surgical Pathology Accession and Grossing” http://grossing-technology.com/newsite/home/perspectives-in-grossing-technology/root-cause-analysis-in-specimen-misidification-in-surgical-pathology-accession-and-grossing/

at the link Perspectives in Grossing Technology.http://grossing-technology.com/newsite/home/perspectives-in-grossing-technology/ The modern bar coding methodology makes the specimen’s correct identification almost a solved problem. There are plenty literature on this subject. For example, PRIMERA company www.primera.com offers equipment that make indeed possible to print very small Datamatrix 2D bar codes which include a lot of data. And what is very important that PTLab Software ties everything together and connects back through most popular LIS systems.

 

 

 

 

 

 

 

 

Bones in the Surgical Pathology Grossing Room

 

Bones are cumbersome and sometimes physically challenging objects in the surgical pathology grossing room. The lack of ability to do bones is often the cause of the processing “misbehavior.” There are some technical details that can make the bone cutting procedures easier and more accessible even to people with minimal physical dexterity.

These remarks reflect my personal experience at the grossing table in surgical pathology. They do not intend to substitute for the grossing manuals, but are an attempt to advise how to best execute the manuals’ recommendation.

The technical aspect of bone cutting is rarely mentioned in surgical pathology manuals. Some manuals are inaccurate by presenting unrealistic illustrations. For example, a very popular Surgical Pathology Dissection: an Illustrated Guide by Hruban  & others shows a cut (dangerous, by the way) of a femoral head, like a wooden board. The goal is to get the most representative section for histology.

A proper bone cut depends on three main conditions: first, firm support, second, an appropriate saw and third, preliminary orientation. The most difficult of these is the secure support of the specimen.

IMMOBILIZATION

Although it is understood that the bone should not move while it is being cut, it not easy to establish a firm grasp of the specimen without compromising convenience as well as an appropriate surface for cutting.

Many devices are used to ensure appropriate immobilization of the specimen, starting with a vice and ending with a variety of contrivances that are offered by manufacturers. Each of them can serve satisfactory in some occasions, but they lack the flexibility for working with diverse bony specimens.

The devices provided by the industry are either flat or round, but bones are, with some exceptions, neither strictly round, nor absolutely flat. These devices also have a second disadvantage as a metallic surface that is undesirable because it prevents from getting a complete section of bone especially with attached soft tissue. Some devices, for example MOPEC’s, make a blind cut, but the section that is intended for histology should be the most representative. Finally, they are bulky and cannot be placed on the grossing station table close to the exhaust and the sink. Even, a relatively small Shandon’s parallel hacksaw measures 44 x 25 x 11 cm. The difficulties of an appropriate cleaning of these devices should also be taken in the consideration.

A variety of self- constructed wooden cutting boards with carved surfaces have been used in research and clinical laboratories. Wood is the most appropriate material for bone cutting support devices.

For many ears, I have used a modified The Davidson Marking System wood holding tray. This stand has various hollow spaces that can be adjusted to different bone surfaces. The pegs sometimes help to navigate the saw as well as serving as additional support.  The significant advantage of this wooden bone- cutting device is that it can be used on the standard grossing station table. It is easy to clean in the sink and convenient for disinfection. Numerous cuts, like a warrior’s scars, reflect its frequent use.

(See BONE SUPPORT GADGET in the link Instruments & Gadgets).

Although a wooden support device is the most appropriate for every day practice, there are situations when it is reasonable to use plastic. For example, I use a Styrofoam box or lid for a longitudinal cut of a femur with attached soft tissue. Some urethane foam plates surround the specimen for a relative immobilization. Even a packaging pressed thick cardboard box (every laboratory has abundant of them) can be used for immobilization in a case of an unusual configuration of a bone specimen.

(See HARD-PRESSED PACKING CARTON GADGETS in the link Instruments & Gadgets).

There are occasions when a bone cut is made on an amputated leg or an arm. Good support is essential, otherwise the sliding and twisting specimen is difficult and dangerous to cut.

I have used a large (32 oz) plastic bucket with semicircle cuts to put the limb in them. The bucket is filled with water for more stability. This simple gadget makes the bone cut, besides other advantages for soft tissue and vessel dissection, effective and safe.

(See SUPPORT FOR AMPUTATED EXTREMITIES in the link Instruments & Gadgets).

The second condition for firm immobilization is an appropriate forceps. The best for this purpose is a Russian Tissue Forceps (203.2 mm) available from MOPEC that grasps all surfaces of the bone. Its firm but flexible grasp allows the user to change both the direction and the angle of the cut, especially in complicated cases involving both tumor and bone. Some gadgets like a meatball maker work in a case of a standard femoral head, but there is no real substitute for a forceps with a toothed dip for a firm hold on the bone during cutting.

A vise remains a reliable tool for bone immobilization. The vise with a vacuum base that is attached to the metal surface of the grossing table is indispensable when it comes to complicated bones like mandible.

SAW

No one can dispute the importance of an appropriate cutting instrument. Obviously, different bones and situation require different kinds of saws.

The Stryker-like oscillatory saw is indispensable in doing autopsies, but it can also used in surgical pathology grossing room, especially if it is necessary to cut a long tubular      bone or a femur head. However, this type of saw has some negative features that are important in surgical pathology. First, the high speed of the oscillating blade requires a more precise approach to of the initial cut. Second, the boney tissue is easily overheated, and sometimes even burned at the site of the cut. Third, the saw generates abundant undesirable bone dust (only diamond saws like the Bueler Isomet Low Speed Saw do not introduce bone dust into the specimen). Fourth, the standard blade cannot be used if the bone is more than   23 mm thick. Actually, an oral surgeon’s Stryker type saws are more preferable in surgical pathology. In general, the Stryker saw requires more training and skills for appropriate use.

The band saw can be used for large and/or dense bones, especially if it is necessary to obtain a longitudinal cut of a slab of bone tumor (for instance, to evaluate tumor necrosis in chemotherapy). However, the space-occupying commercial band saw is more crude, creates more dust, takes more bone material while cutting, soils the room, and more dangerous to personnel (Modern Surgical Pathology, 2003 by Weidner and others even placed a photograph of a band saw injury). I would not recommend it for a standard surgical pathology grossing room, unless it serves a special investigative purpose.

I have read some positive statements regarding band saws, including Mar-Med Band Saw, but my personal experience do not confirms them. Band saws require a reliable immobilization of the bone to make mechanical cutting more efficient for every day use.

The regular handsaw is the best option in most surgical pathology cases. A preferable blade length is 10 “ (the 32T is better for fine cutting, the 14T works faster).  This saw is suitable for a variety of bones beginning with femoral heads and ending with facial bones. It can be used easily on the grossing station table. This saw is easy to clean. The blade is inexpensive and it can be changed frequently.

The fret saw, like the Precision Hacksaw (TOOL SHOP) with a 6” blade is very useful if a fine cut must be made, especially if soft tissue or tumor tissue is attached to the bone. This saw is most useful in cutting maxillary bones, ribs and other fragile bone structures. When there is a small bone or a specific area of interest, the loss of bone material during cutting should be kept in mind. In these situations the thin blade of a fret saw is very valuable. Sometimes, I begin with a Stryker saw and finish with a fret saw, for example, in mandibles with an attached tumor. A fret saw is very useful in larynx with ossification of cartilages.

The low speed diamond saw (Buehler Ltd.) is very useful in cutting a bone with metallic mesh inside that occurs in complicated bone fractures.

There are other useful instruments for handling bones in surgical pathology, for example, curved rib shears, hammers and chisels, but the saw remains the primary instrument.

Using a knife/blade with a hammer, as occurs not so rare, should be discouraged as ineffective and unsafe.

PRELIMINARY ORIENTATION

This part of the bone cutting procedure includes not only decisions regarding pathology issues, but also the correct approach to the area of initial cut to maintain anatomical and pathological relationship. All soft tissue incisions should be made before, precisely at the areas of intended bone cuts. The bone cut should be made through the soft tissue/tumor incision while holding the bone down on the wooden support board. The soft tissue should be taken out quite wide directly at the thickness of the saw’s blade otherwise the soft tissue will be stripped off from the bone by the saw’s movement and the bone will be almost denuded at the places of interest that compromise the diagnostic outcome. This is important when it is long longitudinal cut of the bone, for example in a femur or tibia with a sarcoma diagnosis. This is also important when a rib is cut with adherent soft tissue, due to the fragility of a rib.

Another part of orientation is the determination of the appropriate sequence of cutting to keep parts of the bone available for forceps support otherwise a good section is not possible. The direction of cuts is the main point of the preliminary orientation. Of course, the golden rule of surgical pathology, to make all cuttings in one direction should be honored. Usually, the cut should be made as much possible perpendicularly crossing the longitudinal approach, although there are numerous exceptions. The rib could be mentioned as one of them, if a multiple myeloma is suspected.

SECTION. Of course, the section should fit a standard histology cassette, but it is better if it is at least 1 mm from the borders, otherwise it does not fit the standard embedding mold that is slightly smaller than the cassette. During embedding the section should be easy to orient. Sometimes bones are difficult to cut and it is easier if the microtome knife strikes the periosteum or important structures longitudinately.

The section should be around 2-3 mm thick. Lester’s Manual of Surgical Pathology (2001, page 118) recommends 0.5 cm, but it is wrong because even the depth of the standard histology cassette is less than 5 mm. However, a thin bone fragment (less than1 mm) has a tendency to pop out during microtome cutting. The trimming before cutting uneven surface should also be kept in mind.

The bone dust is undesirable product of bone cutting because the debris is pressed into the bone surface. It is a good idea to rinse the section under running water using a soft brush immediately after cutting.

Some well-known rules that often are not followed.

FIXATION. A common mistake occurs when the entire fresh bone is placed in fixative with the assumption that the bone should be completely fixed before cutting. In reality, even overnight fixation of the entire bone is insufficient, because the fixative penetrates very slowly through the periosteum. The bone should be cut before fixation. However, if soft tissue, especially a tumor is attached to the bone, the initial fixation should precede the bone cut. Then the cut section should be fixed again.

DECALCIFICATION. The sole goal of decalcification is to make the bone suitable for cutting on the microtome. Undecalcified bones are examined for the diagnosis of metabolic bone disease that rarely occurs in clinical practice.

Before discussing the technical details, it is necessary to write in bold:

DO NOT put a bone for decalcification BEFORE cutting and fixation. Everyone agrees but not so many follow. Failure in decalcification can affect all previous efforts by defects in nuclear staining. While decalcification solutions differ in recipe, all of them contain a mineral acid that, in contact with unfixed tissue (soft tissue, bone marrow, etc.) leads to some kind of colliquative necrosis.

 

 

Only fragile maxillary and ethmoidofrontal bones with already diagnosed tumor can be mentioned as an exception when it is better to put the specimen after fixation for decalcification that enables precise cuts with a knife to determine the margin’s involvement. I believe that only very fragile parts of the specimen might be exempt from initial cutting when the appropriate technique is applied.

 First, the section must be completely fixed. Mixtures of formaldehyde and formic acid can provide simultaneous fixation and decalcification, but they are rarely used in practice due to prolonged procedure.

Second, keeping the section too long in decal solution is much worse than under decalcification. Over decalcification is a very common mistake. Under decalcification can be corrected by some measures (the histothechnicians have some methods, like surface decalcification on block).  Over decalcification is often irreparable.

The acid solutions require monitoring the end point of decalcification. The specimens with combination of bone and soft tissue require especially careful decalcification. The end point of decalcification can be tested by the flexibility of the section. Some histotechnicians determine sufficient decalcification   if a fingernail leaves an indentation in the bone section.

Third, the section should be rinsed with running water after decalcification for 10 –15 minutes. Although SurgiPath do not recommend rinsing, it is wrong because hydrochloric acid contaminates the histology tissue processor.

SAFETY. Bones are the most hazardous material in a grossing room due to the dust while cutting and the possible trauma by the instruments or bone fragments. This means that in addition to the universal safety precautions, there should be applied some specific rules.

It is unrealistic to recommend the dust collection devices like the Mopec’s  DCD.

Even in autopsy room they are not used, unfortunately. The Autopsy Bone Saw Extraction System (Surgipath) is inconvenient in surgical pathology grossing room.

Each bone cutting should be done on a grossing station table where some kind of exhaust and running water in the sink is available. Two pair of gloves and a shield mask is obligatory especially if a Striker type saw is used.

A rib is handled differently from a mandible with teeth. The appropriate technique and reliable tools are main conditions for successful bones processing in surgical pathology grossing room.

This  article summarizes the general approach to bone grossing in surgical pathology. More details are presented  in posts at the links “Grossing Techniques” and “Equipment, Instruments, Gadgets”, as well as in my article  Dimenstein IB: Bone grossing techniques: helpful hints and procedures. Annals of Diagnostic Pathology 2008; 12:191-198.

Introduction in Biopsy Grossing Techniques

Selected biopsy grossing techniques cover some areas which are inadequately represented in the surgical pathology grossing literature, including the current main source as the Lester’s manual. All materials reflect my personal experience therefore they have some bias. Although different practices might apply slightly different approaches, these materials try justifying the suggested methodology.

General principles of biopsy grossing techniques are presented in the article Dimenstein IB: Grossing biopsies: an introduction to general principles and techniques. Annals of Diagnostic Pathology2009; 13: 106-113 and in this website’s the Grossing in Dermatopathology manual. However, a standalone description of common principles would be useful for selected biopsy grossing techniques.

Definition of biopsy: the removal for histology examination of tissue structure from living body for diagnostic purposes.

Biopsy grossing starts at specimen receipt. Triage facilitates optimal handling during specimen accession. Besides workload considerations in separation of specimens, there are three main points of grossing triage:

Category (surgical pathology, cytology, microbiology, molecular biology etc.);

Priority (rush, processing workflow);

Preservation (fixation for regular processing, reagents for ancillary studies).

The modern biopsies processing includes many ancillary studies which require different modes of reservation. Fixation is only one though the most ubiquitous technique of preservation.

The table 1 summarizes most frequent biopsy preservations methods although this table is incomplete due to the variety of studies and reagents.

able 1                  Biopsy preservation methods

Type of preservation Reagent/s Purpose Special conditions
Transport isotonic liquid Normal saline (0.85% NaCl) Prevent from drying Short term of exposure due to maceration and autolysis
Fixatives Formalin, glutaraldehyde, Bouin’s solution, Zink formalin,   UMFIX, Prefer, ExCellPlus, etc. Preserve cellular structure, “hardening” for grossing   sampling Minimal and maximal time of exposure, monitoring
Transport medium Michel’s (Zeus) solution Preserve for immunofluorescence Limited time for exposure (2-5 days)
Nutrition medium RPMI 1640 (Roswell Park Memorial Institute)  medium Flow cytometry for immunophnotiping Preferable sterility to preserve antigen integrity
Nutrition medium DMEM-F-12 (Dulbecco’s modified Eagles’s  medium) contains antibiotics and  10% fetal bovine serum cytogenetics Maximal sterility for cell culture
Freezing Liquid nitrogen, isopentan Molecular biology, muscle, nerve biopsies Snap freeze
Transport medium ThinPrep Test Cell preservative solution Prompt use

As a variant of preservation, fixation has a dual role. Besides its main purpose to preserve cellular structures, it serves so called hardening of the specimen that is important for grossing manipulation. Hardening is an old term that stems from pre-formalin era of alcohol fixation. Actually fixation provides immobilization of the specimen’s tissues by preventing them from moving during cutting procedure.

In biopsies when differences between specimens by size and consistency are the most prominent, variations in fixation have practical significance. Formalin is a slow non coagulant fixative. Although the speed of fixation can be accelerated in higher temperature, vacuum and pressure processors, microwave assisted processing, the classic notion that 10% buffered formalin penetrates about 1 mm/hr remains as a guiding point. When a “rush” specimen arrives in formalin, it does not mean that the specimen is completely fixed. Change in color of blood can be accepted as an approximate end point of formalin fixation sufficient for grossing purposes. Formation of acid formalin hematin makes blood turn tan- brown. This is important in biopsies which arrive at different stage of fixation. When the red-pink dots of the areas of biopsy removal disappear by turning tan, the specimen can be considered ready for tissue processor although the fixation is continued in the dehydrating alcohols. Many biopsies, for example needle biopsies, do not have these clues which require additional time for fixation just in case. The current requirement for Her-2 assays of not less than 6 hours is another example of importance to adhere to terms of fixation.

TECHNIQUES in GROSSING BIOPSIES

Sampling, or cut-in, is the main part of biopsy grossing. This procedure requires following appropriate grossing techniques. Biopsies have little space for errors in grossing manipulations. Every action should be precise and decisive.

General requirements to biopsy grossing technique: complete, representative, informative.

                                 

Complete, or entire – all material must be submitted entirely using all means of grossing;

Representative, or adequate – the submission should present the most diagnostically valuable parts of the biopsy specimen for the pathologist’s review;

Informative, or definite – the sample should have clear and unambiguous instructions for further processing during embedding and microtomy.

COMPLETENESS

The fundamental rule of grossing biopsies is in completeness of the submission. It depends solely on the grossing person, especially if the specimen contains numerous fragments or uncountable material. Every biopsy grossing description in the pathology report must include a statement: “submitted entirely.” If technological details are not taken into account “submitted entirely” statement is an empty phrase.

The most popular method to make visible small fragments is to mark them with Hematoxyline or combination of eosin Y and Phloxin B (10:1).

In extremely small fragment, besides marks of warning on the cassette, a procedure that can be called dye crystallization is helpful. By putting layers of Hematoxyline, the minuscule fragment is “incorporated” in dye that is taken away by initial trim by the microtome blade. This method has been tested in many specimens, including on vocal cord or brain miniscule biopsies.

Separation in different cassettes is useful, if size of fragment is dramatically different, especially when a very small fragment accompanies big visible fragments. This often occurs in cervical biopsies.

Completeness of biopsy representation is especially important in breast needle biopsy owing the heterogeneity of the material. Number of cores is substantial because the pathologist should be confident that all cores are examined. Cores should be aligned in parallel on a flat surface during grossing and repeated in the embedding mold. Prostate needle biopsies are another example of heterogeneous core material that requires special techniques to provide completeness of submission for processing.

All generations of biopsy grossing practitioners tried to use different means to provide completeness of biopsy submission. These attempts can be approximately divided in two categories: “gadgets” and techniques (Table 2).

Table 2.    Methods for complete biopsy submission

 

“Gadgets” Techniques
Lens paper wrapping Filtration
Nylon 0.2 mm mesh, paper bags Cell blocks
Foam pads HistoGel, agar-agar
Special cassettes Dye crystallization

The simplest and most available is wrapping the specimen in a cassette size lens paper, but is necessary to mention some details. The paper should be wet, wrapped in envelop like squares with the specimen directly in the center, better circled with a red pencil (Chinese pencil, for instance) especially if the specimen is small. Unwrapping poorly bend paper in not only a pain in the neck for embedding but also valuable material might be lost in folds.

Some laboratories use Obex round papers folded as cones. Tea bags (Salada tea) can be used because they are made of long-staple abaca fibers. Some histotechnologists use Jumbo End Wraps from beauty store (very cheap!), especially for cell blocks. The popular and convenient nylon cloth 0.2 mm mesh diameter bags, as well as porous paper bags, have unfortunate deficiency that the specimen or part of it pops out that can cause loosing valuable material.

Nylon Tissue Biopsy Bags ar offered by Merrick Inc. (Fig.1)

An partially opened Nylon Biopsy Tissue Bag

There are numerous fenestrated mesh processing cassettes which have filtration abilities. They look attractive and in many instances convenient but the problem is how to get the completely submitted specimen in the embedding mold. In opposite to the lens paper and nylon bags when with one movement of a blade the content in the melted paraffin goes in the embedding mold completely, the specimen ought to be transferred from the edges of the fenestrated cassette that is technically difficult. By the way, biopsy cassettes can trap air causing they float.

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Figure 2. Fenestrated cassette .

The fenestrated cassettes are continuing in development of the design. Very interesting option provides Vortex ™ Cassettes. More oval and smooth inside makes easier to embed with assurance of submission’s completeness.

Ubiquitous cellular polyester urethane blue foam pads (can be different colors) are the most popular tools for providing completeness of a biopsy submission in the cassette when small samples are sandwiched between two pads  and placed in a cassette or in tissue capsules. Simple and convenient for embedding, they have some deficiencies as far the completeness is concerned. For example, they are used in prostate needle biopsies, however, the cores in vast majority of cases are different in size and thickness. Some of them can be trapped in the holes of the foam under condition of vacuum and pressure tissue processors. There are observations of artifacts, especially if the biopsy has not been fixed enough. Artifacts, as well as loss of the material occur if the pad is dry or the biopsy has mucinous content. The macro waive vacuum assisted processor’s manuals also object their use for carried over consideration that is a short term processing should be taken in consideration. Again, as little as possible, but they are indispensable in dermatopathology orientation. Hopefully, they will be replaced by other methods in the future.

There are no questions lens paper vs. sponges or fenestrate cassette vs. nylon bags and so on. Everything depends on the concrete grossing situation and specifics of the specimen. However, the foam  sponge should be avoided as much as possible. Actually, I would leave them for orientation purposes only.  Suggestions for Telfa™ pads looks unreasonable technologically because they are not tailored for a processing cassettes, besides that they are impregnated by an untested antimicrobial chemical component.

While working with bags, pads, and special cassettes, in this author’s experience, filtration directly in the processing cassette is the best method if there are either numerous fragments or scarce, especially mucous, materials. The importance of filtration is underestimated in grossing biopsies.

A wet baby diaper pad, as an absorbing material, under the cassette and moistened porous lens paper like fabric inside the cassette can provide effective filtration. This fast method assures complete submission of the biopsy with minimal use of instruments, preventing any contamination. See  “The Diaper Pad for Filtration in Surgical Pathology” at the link “Equipment, Instruments, Gadgets”.

Cell block as a routine in cytology, often is used in biopsy grossing if there is scarce material that can be lost in processing if the standard processing is applied. The most often this method is appropriate if the biopsy is taken with a brush (endobronchial, urethral). Although fixation is an impediment for cell block method, intensive spin can provide diagnostic material.

HistoGel tissue processing medium can be used for complete submission extremely minute specimens, for example brain biopsies. This method now replaced the old method of keeping small biopsy fragment in agar-agar . I do not have an experience with this method but met many histotechnologists who work in neuroscience with positive referrals.

Cucumber method attached small biopsies to squares of cucumber, which had been preliminary dehydrated in several changes of alcohol with a egg-albumin/glycerol mixture. The biopsy/cucumber unit was embedded together after processing and cut and stained as one unit. This method was used rarely for cervical biopsies (very small), but more frequently for gastric and especially duodenal biopsies. It looks that now it was abandoned. In general, the method is good, though cumbersome, but in practice, often cucumber and the biopsy arrived in one container but separate that made the idea of orientation by the clinician a mockery, although this could be fixed by a grossing person who understand the process. In principle, this method can be used, although there are other organizational issues. I doubt that this method can be used now in the assembly line biopsy production.
Transfer instruments and devices Although in the vast majority of biopsy grossing situation the forceps, or different kind of them depending on the size of the specimens and conditions, are used in transferring biopsy into processing cassette, there are situations when other instruments or devices can be employed. The ideal would be direct filtration from the container into cassette that most appropriate in endometrial, cervical curettage, or bone marrow aspirate, but even in this situation sometimes forceps is used to assure completeness of submission.

In a case of numerous fragments, as in gastrointestinal biopsies, a disposable transfer pipette, like S/P Plastic Transfer Pipet, is cheap, fast and reliable. In rare occasions when the film like specimen is difficult to handle, an inoculating loop or nichrome wire loop like device can be used for reliable transfer of the specimen from the container into cassette (of course, using the lens paper to wrap up).

Completeness of submission depends also on clinicians who send the material. The common habit, for example, to send curettage samples on napkins is wrong. Clear product should go to pathology. Some napkins deteriorate in normal saline, as well as in formalin. The specimen slides to the fringes, steaks there. Under these circumstances, it is difficult to recognize the specimen and to take it out.  Endometrial curettage as well as vocal cord polyps in SafeTouchTissueTrap cannot be accepted as biopsy. It is difficult, almost impossible to get out all material from the holes of the container. Now colonoscopy practice uses often Suction Polyp Trap to collect polyps in capture chambers with perforated bottom, and some of the material sticks in the perforation so tight that there is no way to take them out without damaging the tissue. And, of course, already mentioned mode of sending material in dry condition is the path to lose biopsy material.

Extraneous tissues, or floaters

The completeness of submission has its opposite as extraneous tissues, “floaters.”  In biopsies floaters might be not only a nuance but liability under condition of modern surgical pathology with numerous very much similar biopsies. Grossing has it significant share as culprit of this unfortunate error of processing. A set of preventive measures (cleaning and changing the processing board, instruments, and gloves) must be implemented. Floaters problem requires a comprehensive approach starting from specimen receipt to histology stainers, but grossing is one of the main areas of contamination that might have significant diagnostic consequences. Details of preventive measures are presented somewhere else. See “ Extraneous Tissues in Surgical Pathology Practice” at the link “Perspectives in Surgical Pathology”.

REPRESENTATIVE and INFORMATIVE

Both of these components of grossing are inseparable. Ink and knife are means for nonverbal communication between the grossing person and the pathologist, as well as the embedding person and histotechnologists.

INK

If the ink does not carry any message it does not make any sense to ink. Ink can obscure the pathologist’s vision. In thin specimen insoluble ink fragments can diffuse from the area of inking. In all questionable situations it is reasonable to abstain from inking.

What can the grossing person signal with the ink?

          1/ Margins to the pathologist.

                                              2/ Specific areas of interest to the pathologist.

          3/ The mode of embedding.

Margins are a questionable issue in a small biopsy. Some dermatologists are resentful if a pathologist mentions margins in a shave biopsy, unless they specifically ask for. Nevertheless, grossing person should provide the pathologist with this option in the report. Ink obviously makes it possible.

The main goal of inking in biopsies is to instruct the embedding person about the mode of embedding. If the specimen were cut, it is appropriate to ink because the section area remains unstained. This area should be put down in the mold during embedding. If the specimen were not cut, the inked area should be on the periphery. For example, an inked dot at the subepithelial area in a cervical biopsy gives the embedding person definite instruction that during embedding the dot should be at the periphery (Figure 3). Otherwise, the most valuable epithelial material can be lost or invisible on the slide.

Ink can point to specific areas of the specimen’s special interest for the pathologist. For example, area of merging of ectocervix and endocervix in cone biopsy (Figure 1).

Figure 3. Two color inked cervical cone biopsy

Details of inking technique

There are some technical details that make inking efficient or … confusing. They can be summarized as a/ choice of ink, b/ application, and c/fixation of the ink.

Davidson’s Marking System dyes are the commonly used in laboratory practice. Although the color of ink is often a personal preference, there are some considerations in choice of a dye. For example, green almost never interfere with slide stain colors and always visible during embedding. Black ink (India ink in art shops) is protein based that is an advantage because it can be more secure “fixed” to the tissue by cross-linked fixatives; this is  also disadvantage for fast processing because the dye is prone to wash off if there were no time for exposure to the dye. Although multicolor inking is rarely applied in small biopsies the rest of dyes can be arranged in the order of less wash off: orange, red (merbromin), yellow( except adipose tissue, but not in lipoma due to the capsule), blue (common laundry bluing).

I have read about tattoo ink as a marking tool, but haven’t seen any facility, which used them. I do not think that they will be cheaper than traditional vendors charge if you by small quantities. However, the main thing is that we do not need an intrinsic involvement with the inked tissue. Surgical pathology uses margin and orientation marking inks. Tattoo ink is intended to stay forever, for some users unfortunately.

There is a variety of dye applicators from cotton tipped (rarely used in biopsies) to wooden picks. In biopsies, precise dye application is a priority. It seems that cotton tipped stick are not appropriate completely in biopsies because they are not precise and have threads which can be seen under the microscope give additional difficulties to the pathologist.  In the author’s experience, that flat angled .17 mm smooth nonwettable Stratagene’s Strata Tips are the most applicable for inking biopsies although other similar applicators can be used (Figure 4).

Figure 4. Plastic ink applicators

A common practice is to use a “fixer”, or color enhancer, which is incorrectly called mordant (histology definition of mordant is a lake of stain between reagents). Most mordants contain acetic acid, for example, in concentrations of 3% or 50% of white vinegar. In Bouin’s solution acetic acid is a component. Some institutions use acetone but it is undesirable for many reasons, including safety considerations. The ‘fixer” can be applied in different ways, such as using a pipette or sponges (foam polyester urethane to non-latex make-up sponges) after the dyes have been applied.

The dyes are more effective when applied to unfixed tissue. Fresh tissue should be patted dry. The common pattern includes: dry vigorously until the paper is not bloated (underestimated step in practice), apply the dye, air- dry for a while, apply a fixer carefully, and dry gently with a sponge, then process.

KNIFE

To cut or not to cut?

           Do not cut if

the section will be less than 0.2 cm (difficult to embed straight);

the area of diagnostic interest is too small (less than 1mm) because it might disappear after section (the cut takes  tissue and microtomy trimming also);

unfixed specimen less than 0.4 cm, especially with soft/adipose tissue.

Three main conditions are required for taking correct biopsy sections:

1/ Uniform sections –desirable, but impossible without special devices;

2/ Representative – possible, requires understanding pathology issues and ability to present the most diagnostically valuable section;

3/ Informative for embedding- obligatory for the ultimate goal to provide the pathologist with representative pathology material.

A uniform thickness of the section is an obvious requirement for processing but it is difficult to achieve it under conditions of uncertain immobilization of the specimen. The microwave assisted accelerated processing put more demands on uniformity of section. Special device can make uniform 1.5 mm section for X-press microwave assisted processing . The CutMate forceps introduced by Milestone is step ahead in attempts to get uniform preset thickness sections, however, it is completely useless for biopsy section.

A representative section is possible if the grossing person understands diagnostic goal/s of the specimen though not always possible due to variety of the appearance of pathology and some technical conditions of manual section. Sometimes uniformity of section should be sacrificed to avoid unrepresentative section. For example, in a squamous cell carcinoma vs. seborrheic keratosis the section of often fragile lesions should be presented in full that is unachievable without making thicker sections. Or adenomatous villous colon polyp requires special technique of sections to preserve integrity of the specimen by maintaining the relationship with the stalk in most informative way.   Or a cervical cone biopsy requires certain cutting technique to present to the pathologist sections that reflect ecto/endo cervical transitional areas.

An informative unambiguous section prevents the embedding person from guessing how to put the section in the mold without sacrificing the informative material. For example, if the section has both, the area of diagnostic interest and adjusted tissue without such material, the embedding can be wrong by concealing from the pathologist informative material. Figure 5 illustrates a wrong section when the pigmented area is only from one side of the section.

Figure 6. Ambiguous section (the pigmented area is only from one side)

Orientation techniques

Ink and knife/blade do their part in presenting the most informative diagnostic sample. The ultimate goal of grossing is correct diagnostically sound specimens/sections orientation.

There are different techniques that can be used for proper orientation. Some of these techniques belong to history, some remain in practice. Periodically some forgotten techniques are brought back with corrections.

Many techniques have been employed foe embedding orientation of biopsies. One technique involves sticking a specimen to slabs of Gelfoam with cyanoacrylate. A different technique arranges a duodenal biopsy mucosa upwards on small strips of ruled cellulose acetate. Agar-agar has been used for orientation of biopsies and small specimens. Dehydrated slotted or plain cucumber slabs have been initially recommended to mount cervical but now  remain predominately for gastrointestinal , especially duodenum , sometimes for laryngeal biopsies .

HistoGel tissue-processing medium can be used for specimen orientation, for example brain needle biopsies.

Two foam polyester pads used to sandwich the specimen in a position for embedding. This method is very popular, however this often does not help the embedding person to correctly orientate a biopsy, especially if it is too small or of uncertain shape. Sometimes the specimen sticks to the upper pad or pops out when the cassette is opened.

A rarely used method of superficial incisions in spongy pads was proposed for small biopsies. Complete through the pad incisions can make this method more universal, especially for orientation skin, cervical, and gastrointestinal polyps. The figure 7 presents orientation of a biopsy.

 

 

 

 

Figure 7. Biopsy orientationorientation in the foam pad incision

 

The search for methods of specimen’s orientation continues. There are proposals of cassettes with fluoropolymer section able immobilizing porous platforms or silicone pads for embedding orientation, but they are still at the stage of testing.

Correct embedding orientation is the last line where grossing technique can directly influence the specimen processing. The rest is in hands of histotechnologists although wrong grossing technique like poor fixation, wrong thickness of the section, and incorrect placement in the processing cassette could compromise cutting and staining process.

The general principles and techniques will be repeated in surgical pathology subspecialties (gastrointestinal, gynecology, etc.). Dermatopathology is presented in a separate manual  “Grossing in Dermatopathology.” There are specifics in details of grossing in every subspecialty that should be addressed.  Selected biopsy techniques will be presented in separate postings. They reflect personal experience.

Fixation, Fixation, Fixation

 

.                                                                   “Oh stay! You are so beautiful!” J.W. Goethe, Faust

INTRODUCTION

Is histotechnology art or artisan? This question sparks discussions among histotechnologists. Most lean to the assumption that histohechnology is clearly artisan that can be performed with artistic elegance and beauty.

In artistic terms histology is closer to “still- life” or in French “nature morte” (literally dead nature). The flowers are plucked, but the goal of the artist is to stir up imagination of their scent.

Histotechnology has a more modest goal as to help the viewer, in most instances the pathologist, to generate an informed conclusion, namely the diagnosis. The real beauty of histology is in maintaining the immediate pre-removal structures. We have to kill the cell and the tissue to keep them like alive. The key word is LIKE not As. The process is called fixation. It tries to stop the moment. Appropriate and adequate fixation in surgical pathology delivers the beauty of complexity and variety of nature for diagnostic purposes.

Beauty, however, is within the eyes of the beholder. Beauty per se in histotechnology is not valuable if it distracts attention from the cognitive process. That is why pathologists, as consumers, seem to be somewhat unappreciative of histotechnologists artistic work. “Histochemistry: a case of unappreciated beauty?” wrote René J. Buesa on the pages of Journal of Histotechnology.1 The pathologists do not have time and desire to watch even an extraordinary beautiful slide if it is in contradiction with his/her image at the occipital lobes of their brain that allow them to make the diagnosis, sometimes by intuition. Now intuition is more substituted by IHC markers but cannot be dismissed completely. From practical point of view the main value of a histological slide is in the standard consistency of a descriptive microscopic picture. Complete fixation is the key for standard specimen processing.

With the assumption that readers of this website are familiar with the theory of fixation presented in many fundamental handbooks 2,3,4,5,6 , as well as in some theoretical articles.7,8 these materials are intended to offer a pragmatic approach to fixation, predominately in surgical pathology. In a loftier sense, the article aims to express unconventional approaches to fixation process based on the author’s experience and observations, though some issues, and among them safety question, require separate more substantiate argumentation.

Fixation as preservation

Many processes start immediately and simultaneously after the tissue is taken out from the body. Some of them are presented on the diagram 1.

Processes after tissue removal                              Diagram 1

Drying out is the first concern. In mucinous tissues or film like biopsies, for instance laryngeal, it is of great importance.

Autolysis due to the continuation of the enzyme metabolic activity and release of lysosome’s digestive enzymes starts immediately after tissue removal. Some tissue with intensive enzyme activity, for instance, small intestine, pancreas, tubular area on the kidney, can be severely damaged.

Decomposition by infection (bacteria, fungi) is a reality of the environment, although putrefaction and decay do not occur in practice due to the short time between the specimen removal and processing.   In general, the contemporary requirement to tissue processing leans to maximal sterility, or better to say minimal contamination, especially keeping in mind that some ancillary studies (cytogenetics, flow cytometry) use tissue culture methodology.

Fixation stops everything. This was the main rationale for the immediate pouring fixative. However, the classic notion to start fixation as soon as possible after the tissue is removed can interfere with modern variety of ancillary studies which broadens the definition of preservation. In practical sense, more frequent use of refrigeration and sometimes even snap freezing (for enzyme profile) ought to be implemented. As the condition of specimen preservation, knowledgeable pre-processing triage is the requirement keeping in mind the main medical motto: “Don’t do harm.” In this instance, do not impede the possibility of the best diagnostic outcome for the patient.

Dual goals of fixation

As preservation procedure, fixation is aimed to achieve two goals: maintain as much as possible organ/tissue integrity and cellular/extracellular substances structure that existed before the removal procedure.Fixation provides preservation of the integrity and morphology tissue and cells keeping in mind that histology processing includes many “unpleasant” for them physical and chemical processes like dehydration and clearing, as well as embedding and staining to some degree. These goals are presented on the diagram 2.

Dual goals of fixation                                                         Diagram 2

Perhaps, the word protection requires quotation marks. It would be appropriate to define protection of antigenicity as not masking (messing up), as much as possible the tissue protein’s epitope as the immuno-determinant region on the antigen surface to be recognized by the antibody’s binding site. Immunohistochemistry changed the game. By the way, Zeus (Mishel’s solution) transport media does preservation for immunofluorescence, as RPMI for flow cytometry or DMEM-F-12 for cytogenetics.

Fixation for grossing processing

Preservation of the tissue/organ integrity is one of the goals during grossing, or sampling, or cut in, depending on the terminology that is used to define the initial stage of processing. It does not matter if the tissue/organ were cut or not, but, of course, if the tissue were cut, the integrity of the tissue/organ should be the primary concern. The tendency of specimens sampling in fresh state is wide spread due to the pressure of turnaround time. Insufficient fixation prior to sampling is underestimated in current surgical pathology practice.  This is the reason that this article discusses fixation for grossing processing in detail.

It would be an anatomical triviality to state that in opposite to more or less homogeneous cytology material tissues/organs are combination of heterogeneous types of histological components that are in a loose connection with each other. Every intrusion changes these original connections. The goal is to minimize them during grossing when the tissue/organ has to be adjusted to procedural means (optimal presentation of areas of diagnostic interest, size of the cassette, convenience for embedding, etc.). Grossing manipulations move these tissue conglomerates by pulling, pressing, twisting during the manual process.

Fixation provides immobilization of tissue structures for grossing manipulations. JA Kiernan puts it as spatial displacement of structural proteins and other constituents of tissue.6 This process has an old commonly accepted definition, of tissue hardening.  It stems from the dawn of histology before the formalin era when alcohol fixatives hardened tissue.  Alcohol really hardens the tissue frame by dissolving lipoid structures and coagulating protein fibers. Hardening in the early days of histology technique served to improve micro section. As Frieda Carson wrote: “…since much of sectioning was done freehand. Today, with an array of embedding media to choose from, this hardening action is less important.” 5 However, the old macroscopic tactile term “hardening” approximately explains the immobilization of tissue structures for grossing. It is widely used and mutually understandable.

Why do we need “hardening”? Let’s take a simple analogy. The sharpness of the knife/blade is useless to cut a soft bread loaf just from oven. Bread only looks homogeneous, in reality it consists of threads of baked dough. The tissue treads are following the knife pulling the dough under the weight of the knife. If the loaf is not fresh the cutting is easier. The surgical pathology tissues are definitely heterogeneous with loose connections between adjacent structures. The goal is to minimize their relative “movements” towards each others during cutting tissues which are not “fresh”, namely after fixation .

Perhaps, to some degree colloquial, the term “hardening” will stay until a different definition can be found. While using it we always should understand that it is an immobilization method for grossing manipulation to prevent literally tissue/organ mutilation. Hardening contributes to uniformity of section. Fixation in this instance is a “tool” that equalizes deficiencies of grossing tools (knives, blades, forceps, etc.). The more fragile are tissues the more careful and complete should be fixation for grossing immobilization, e.g. “hardening”.

Hardening during fixation comes with a price. In some rare instances, fixation makes grossing more difficult, if the tissues are completely different in consistency. For example, bone and adjacent soft tissue lose natural adhesiveness because the soft tissue becomes more brittle. It is difficult to maintain the integrity of the specimen if grossing were done on the specimen after fixation. Even the finest saw tears soft tissue from the bone due to the principle of sawing. In this situation the pathologist cannot answer the main clinical question about tumor and bone relationship. Working with complicated bone specimens, especially after facial-maxillary surgery, this author recommends sectioning bone and adjacent soft tissue before fixation.9

It seems that lobulated colon polyps fall apart easier after fixation. Eye ball is better to gross in fresh state due to extreme heterogeneity and fragility of tissues. Those are exceptions from the general rule that grossing is more efficient after fixation.

Change in color of blood can be accepted as an approximate end point of formalin fixation sufficient for grossing purposes. Formation of acid formalin hematin makes blood tan brown, although some other conditions (change in acidity can also make brown colors). This is important in biopsies which arrive at different stage of fixation.

Fixation as cells and extracellular substances stabilization

The main goal of fixation is cells and extracellular substances stabilization. Fixation also serves the aim of making the intimate structures more visible by opening channels for staining (5) through complicated physical and chemical processes such as electrical conductivity, viscosity and solubility gradients of reagents. These goals ought to be carried out without masking antigenicity as much as possible.

By essence, fixation uses chemical process although some physical methods are employed, especially with the development of microwave assisted processing. Actually, microwave itself is not more than heat coagulation if the fixative penetration were not completed. Coagulant (alcohols, mercury, zinc, etc.) as well as non-coagulant (formaldehyde, acetic acid, etc.) fixatives interact with proteins by stabilizing them. The coagulant alcohols do not establish a chemical bond with proteins that make them attractive for immunochemistry. The non-coagulant acetic acid with its fast penetration preserves nucleoproteins and prevents shrinking.

Every histology textbook contains numerous recipes of fixation solutions which actually are not used now in practice. Complicated fixative solutions have disappeared gradually leaving only a few of them, just not as a common practice. Among them Bouin’s solution (this author’s favorite for many years, despite many months of ulcers on a finger caused by picric acid), Carnoy’s, B-5. The latter almost everywhere is replaced by Zn formalin or even formalin. Disappearance of complicated recipes has many reasons. Among them are, in the author’s view, environment considerations (mercury-Zenker, B-5), the necessity to ex tempore mixing ingredients, improvement of technology processing (VIP, microwave), proprietary stock solutions, the necessity close monitoring, …and, yes, lowering the bar of requirement to quality of microscopic picture owning in part to the help from IHC markers.

Two main groups of fixatives are in competition at the practical laboratories market: formalin and alcohol based solutions, although there are, of course, mixtures of them.

Formalin

Fixative recipes come and go. Formalin has stayed for a long time, although during Virchow’s time, in the 19yh century, it was not in use. Formaldehyde is a gas methanal that dissolves in water at a maximum concentration between 37-40% vol./vol. The 10% solution formaldehyde contains between 3.7 and 4.0% of formaldehyde and is called “formalin” in many countries and “formol” in Europe.  Formalin’s formula changed also, but the main idea of NBF (normal buffered formalin), as 10% solution, is ubiquitous. Now a company Azer Formagel even offered 10% Gel Neutral Bur\ffered Formalin with a brand name Formagel. The 7-15 % methanol added as a stabilizer not only delays formaldehydes polymerization but brings some positive fixative features of alcohol. Formalin stored in a cold place usually contains less than the 37% of formaldehyde stated on the label. The missing formaldehyde is in the white precipitate (which is paraformaldehyde) in the bottom of the bottle. Good structural preservation for electron microscopy is possible in solution made from formalin with optimal pH and salt concentrations. The small amount of methanol in a neutral buffered fixative made from formalin could have no fixative (coagulant) action. 10% NBF is not the same thing as 3.7-4%formaldehyde. Whether they can be used interchangeably has to be validated.

Details of formalin fixation are presented in the Buesa’s article “Histology without Formalin?” 10 The article provides a comprehensive approach to three sides of formaldehyde action: penetration, covalent binding, and cross-linking (actual fixation). These work simultaneously but at very different rates: penetration twelve times faster than binding, which in turn is about four times faster than cross-linking. Due to fast penetration, the thickness of the gross section does not matter substantially in the range between till 4 mm (the maximal depth of the standard processing cassette), but temperature at the rate 37-45C (definitely below 60 C) can accelerate fixation. By using laboratory microwave oven accelerated processing with temperature to 50-55 C, the time of fixation can be substantially below the standard 24-48.

Zinc formalin fixative is accepted in surgical pathology practice for better nuclear details, especially in bone marrow biopsies. As with B-5, it requires monitoring. The time of fixation is 1.5-2 times longer. Zinc formalin is praised as immunochemistry friendly fixative because it often made antigen retrieval unnecessary. Anatech Ltd developed Z-Fix, a buffered zinc formalin that prevents formalin pigment while maintaining IHC fixative quality.

The formalin disadvantage is incomplete DNA and mRNA recovery. Formalin is also unfriendly towards molecular biology. Two disadvantages are the slow pace of fixation (24-48 hours at the room temperature) and necessity for heat induced antigen retrieval to overcome the deleterious effects of formalin fixation. All these disadvantages are reasons for the search of a different fixative.

Alcohol fixatives

Actually, fixation always is finished by alcohols while processing for paraffin embedding if cross-linking were not completed before. The process of dehydration is part of the final fixation.

The main alcohol fixative that survived since 19th century is Carnoy’s fixative. Originally it contained 100% or 95% alcohol (6 parts), chloroform (3 parts) and acetic acid (1 part). All old timers can remember it as a pain in the neck (problems with absolute alcohol, evaporating narcotic chloroform, ex tempore preparation). However, the nuclear result was excellent. Then chloroform was eliminated (Clark’s fluid), and ethanol was substituted by methanol (6 parts). Methacarn replaced Carnoy fixative in practice with good results for fast penetration and nucleic acids preservation though it is not recommended for most surface antigens.

Methacarn is only one of numerous alcohol fixatives to name some of them (OmniFix, NeoFix, BoonFix, EthMeth, F13, MicroFix, UMFix, Uni-Fix, Fine Fix, etc.). The content of many of them is not disclosed. One of them UMFix has an ambitious name as Universal Molecular Fixative that is claimed as a molecular-friendly alcohol based fixative that preserves morphology.13  All these fixatives as well as non-alcoholic formalin substitutes ( Anatech’s Prefer, Preserve, Safe Fix II, NOTOXhisto, Glyo-Fixx, DMS, HistoCHOICE, etc.) are discussed in detail in the above mentioned article “Histology without formalin?” 10  Glyoxal, the active ingredient of Prefer, a promising alternative to formaldehyde. forms crosslinkages longer than formaldehyde. Prefer contains also a small amount of ethanol that helps in penetration through lipids. However, Prefer  has been more than a decade on the market, but still is not accepted by general practice.12

A combination of alcohol and less than 10% formalin, for example IBF (methanol and 2-propanol with 3% formalin) is an option for some biopsies (prostate). There are other low percentage formalin fixatives as S.T. F, Pen Fix, Stat Fix, actually mixtures with alcohols and other undisclosed components.

A buffered alcoholic formalin, CBA Formalin, is manufactured by Anatech Ltd www.anatechltdusa.com. This fixative has the advantage of formalin’s fast penetration as a small molecule and fat tissue dissolving by alcohol, that increases penetration through fatty thick sections. Before the microwave assisted technology, the combination of 10% buffered formalin and 70% alcohol was my favorite fixative. it seams that Anatech’s Alcoholic Z-fix Concentrate ( mixture with Zink formalin) is right , but unnecessary in large specimen, but for bone marrow and similar small specimens (lymph nodes) it is enough  Zn formalin because fatty part is taken out during gross sampling.

The amount of brand names of formalin substitutes is impressive. The list of them is incomplete. Who can even remember them, never mind to have informed conclusion of their quality? Sporadic articles about proprietary (some of them do not have a disclosed formula) fixatives with contradicting always positive recommendations cannot be used as a credible guide for practice.

Where to swim?

The ship of practical surgical pathology histotechnology, with a crew empowered by IHC, navigates its course between Scylla and Charybdis. Histology practice is facing the monster of enormously increased amounts of slides and the pressure of turnaround time while trying to avoid sinking in the whirlpools of untested commercial fixatives offers. There is an urgent call to determine the course in regard to this crucial component of histotechnology quality as fixation.

The CAP regulations, or recommendations or guidelines, regading fixation for the Her2/ER/PR validation change the game because it is necessary to have comparible results between different laboratories for breast cancer treatment. The bread loaf gross sections and sections in the cassettes are not allowed to be processed with less than 6 hours in the cassette plus processor time for formalin.  It is a rule if the breast is not in cassettes before 3PM it cannot go on the processor.   In most cases the breast is fixed overnight, loafed.  Then the sections are left in the cassette for processing within the 48 hour. maximum for Her2Neu.  If they are late on Friday we process Saturday and take the tissue off on Sunday so we bent no rule for the formalin fixation.
The use of any other fixative require a complete validation of the alternative fixative to prove results are identical with the alternative fixative compared to formalin-fixed tissue. However, formalin is the only FDA approved fixative for  Her2/ER/PR.

There is a necessity to conduct a serious, perhaps international (we are not alone in the world), pilot projects under the auspices and direct involvement of a major group, like NSH or ASCP, or CAP, to determine the commonly accepted fixative in practical surgical pathology leaving experimentation to the research facilities. This project should take in consideration many often contradictory issues. Among them are: universality in practical laboratories, minimal masking for IHC, cost, speed of fixation, molecular biology friendly, and, of course, safety. Grossing needs (hardening) should be taken into account. Although there were attempts to conduct workshops, for instance by CAP, orientated predominately for IHC, the problem requires a comprehensive solution.

Keeping in mind commercial and research interests, it would not be a simple task to reach a pragmatic consensus. However, despite clashes of personal preferences, commercial interests, and politically correct tendencies, this task should be carried out. This is for the sake of standardization of histotechnology as a major player in the diagnostic process.

Here are some notions which might be taken into account. In a sketchy form, they reflect how the author understands the multilateral approach to the problem.

Universality as much as possible for the use in practical laboratories is an urgent necessity. In opposite to napkins or greeting cards when diversity is a virtue, fixatives in surgical laboratory ought to be ubiquitous. Each fixative makes artifacts, or aberrations, in the microscopic picture. There is common and individual experience used to evaluate them. Many decades of experience of using formalin fixed microscopic image cannot be thrown out the window.

Minimal masking of antigenicity for IHC is a serious advantage of every fixative due to the crucial importance of antibody markers in the surgical pathology diagnostic process. Noadditive noncoagulant fixative would be a dream fixative. The more realistic option would be a noadditive coagulant alcoholic fixative with its disadvantages of shrinking and hardening. Modified methcarn (8 parts of methanol and 1 par of acetic acid) looks promising. There is a long way to the more or less commonly accepted IHC friendly fixative.

The cost of the fixative is not an irrelevant issue. Laboratories are also participants in the sky rocketing cost of medical services. The low cost of formalin is determined not only by simplicity of production, but also by the amount of demand that drives the cost down.

The speed of fixation is an important factor in choice of a fixative. Turnaround time (TAT) is a reality in the surgical pathology laboratories. It is unclear why the prostate biopsy should be reported the next day (some laboratories try to report the same day) if the patient, as a rule, were scheduled to see the doctor in a couple weeks. Everyone who works in the practical laboratory knows how often supervisors try to please the pathologist accelerating the processing. However, the rush epidemic is a fact of medical life. At least, fixation ought to be complete.

Molecular- friendly is also more a wish than a reality of every day practice in surgical pathology. No any of molecular fixatives can in this point meet main requirements to the universal fixative as cost, simplicity of the recipe, stability in storage, previous experience, the gold standard in Juan Rosai’s words, of generations of pathologists, who will not trade it at this time the elusive gains for diagnosis of molecular biology, even an individually targeted cancer treatment.14

Safety considerations are the most persuasive in arguments of fixative choice for understandable reasons. Formaldehyde is the primary target of safety concerns. Without any doubt it is chemically active. It irritates eyes, mucosa, and skin. This author had had two formalin burns of the eyes, loss of voice after cleaning up a formalin spill, and many other chemical injuries. There are people with allergic reaction even to minimal concentration of formaldehyde in the air.

Among objections to formalin use, the major is formaldehyde’s cancerogenicity. However, the data about formaldehyde’s cancerogenicity are limited.15 Analysis of National Cancer Institute cohort studies presented the excess of nasopharyngeal cancer among workers in formaldehyde industries although data about formaldehyde exposure as a cause of leukemia and lung cancer are inconsistent.16 The safety question requires separate sober discussion. The question of formalin safety is discussed in detail in the article “A Pragmatic Approach to Formalin Safety in Anatomical Pathology”, as well as at this website (see the article at the link Safety in Grossing Room).17

Meanwhile we have to work with formalin. Our stern, but true friend will serve practical surgical pathology in foreseeable time using all reasonable safety measures. Not optimal but merely a compromise tissue fixative.

CONCLUSION

The problem of a pragmatic consensus fixative for surgical pathology remains. Appropriate fixation is the main precondition for a representative, informative, and standard microscopic picture under the pathologist’s microscope. If fixation had been done properly, deficiencies of processing are repairable in most instances.  As in real estate, the motto is: Fixation, Fixation, Fixation.

Acknowledgment

Many thanks to René J. Buesa BS, HLT (ASCP) for valuable comments.

References

1. Buesa RJ., Letter to the Editor. Histochemistry: a case of unappreciated beauty? J Histotechenol  26 (4):283, 2003

2. Pearse AGE. Histochemistry, 4th ed. 1980 Vol 1.Baltimore, Williams&Wilkins Co

3. Lillie RD & Fulmer HM 1976 Histologic Technique and Practical Histochemistry 1976New   York: McGraw-Hill, pp.52-53

4. Bancroft JD, Gamble M: Theory and Practice of Histological Techniques. 5th EdNew York,NY, Churchill -Livingstone, 2002.

5. Carson FL, Hladik Ch: Histotechnology: A Self-Instructional Text 3nd Ed. Chicago, Ill ASCP Press; 2009 pp 1-23.

6. Kiernan JA Histological and Histochemical Methods, 2008, 4rd ed. Oxford: Butterworth-Heimann.

7. Dapson RW Fixation for 1900s: a review of needs and accomplishments, Bioth & Histochemistry 68 (2) 75-82, 1993

8. Wenk PA Fixatives: Coagulatives vs. Non-Coagulative or Is it Additive vs. Non-Additive? Histologic December 39 (2): 36-39, 2006

9. Dimenstein IB: Bone Grossing: Helpful Hints and Techniques Annals of Diagnostic Pathology, Vol12 (3):193-198, 2008

10. Buesa RJ: Histology without formalin? Annals of Diagnostic Pathology Vol 12 (6):387-396, 2008

11. Dapson RW Formaldehyde: Past, Present & Future 2001 HistoLogic May; 34 (1): 5-8, 2001

12, Dapson RW: Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval. Biotech & Histochem: 82 (3) 161-166, 2007

13 Nadji M, Nassiri M, Vincek V et al: Immunochemistry of tissue prepared by a molecular-friendly fixation and processing system. Appl Immunohistochem Mol Morphol, 13: 277-282, 2005

14. Juan Rosai, Why microscopy will remain a cornerstone of surgical pathology Laboratory Investigation, Volume 87, May: 403-408, 2007

15. Formaldehyde (CASRN-50-00-0) Integrated Risk Information System US-EPA, IRIS Summaries.

16. Hauptmann M, Lubin JH, Stewart P.A, et al: Mortality from solid cancers among workers in formaldehyde industries. American Journal of Epidemiology, 2004; 159(12): 1117-1130.

17. Dimenstein IB.: A Pragmatic Approach to Formalin Safety in Anatomical Pathology”. LabMedicine, 2009; Vol 40, (12): 740-746.