CHAPTER 1 Tissue Procurement, Processing, and Staining...

CHAPTER 1

Tissue Procurement, Processing, andStaining Techniques

Mark R. Wick, M.D.,

Nancy C. Mills, H.T., QIHC (ASCP), and

William K. Brix, M.D.

It is an unfortunate reality that many pathologists haveonly a rudimentary knowledge of the effects of surgicaltechnique and tissue processing on the final results thatwill be obtained in stained microscopic sections. All toooften, one is faced with a sample that has been obtainedcrudely, fixed badly, or mishandled in the histology labo-ratory, making morphologic interpretation of it needlesslycomplex. These faults typically occur not through willfulneglect of proper methodology but rather because ofignorance of the sequence of steps that constitute the scienceof histotechnology. Most trainees in pathology are not wellversed in the details of this laboratory discipline, makingthem totally dependent on the expertise of their techni-cians, or the lack of it.

Accordingly, this chapter will provide an outline of rec-ommendations for the procurement and subsequent han-dling of histopathologic specimens. Potential reasons forpoor results are also included.

BIOPSY TECHNIQUES

The specific procedures that are used in performing biop-sies of clinical lesions are usually left to the discretion of theattending clinician. This provision is not a problem if theoperator has been adequately educated on the specificadvantages and disadvantages of various techniques, as ap-plied to specific diseases. However, it may prove to be a di-saster if the surgeon is inexperienced in such matters.Conventional or enzyme histochemistry, immunohistol-ogy, or electron microscopy—all of which are greatly af-fected by nuances in tissue preservation—may be necessaryin some instances to obtain a firm diagnosis. Because theclinician may not be able to anticipate these possibilitiesbefore obtaining the tissue sample, a predetermined rou-tine should be followed in doing so (1).

There are basically four generic categories of proce-dures that may be used in any given case. These includepunch biopsies, using circular cutting devices of several

sizes; partial or complete excisions done with a scalpel;electrosurgical excisions; and laser-mediated biopsies. Inchoosing one of these options, the operator should be cog-nizant of the two opposing ‘‘forces’’ that affect his or herfinal decision on this matter. On the one hand, the patientis often preoccupied with the cosmetic effects of a biopsy,and this typically induces the surgeon to limit the size ofthe sample as much as possible. The opposing considerationis represented by the degree of difficulty with which themicroscopic diagnosis is made by the pathologist—a factorthat is often predictable by the amount of material that willbe required to study the disease process adequately.

The cardinal rule to be remembered on this topic is thata properly done biopsy is virtually never cosmeticallydeforming, if it can be accomplished in an outpatient set-ting by a competent operator. In contrast, specimen inad-equacy and artifactual changes in tissue are problems thatrelate to faulty procedure, and these account for the greatmajority of diagnostic obstacles that pathologists encoun-ter. There is nothing quite so aggravating for the clinician(and the patient) as to be informed that a second biopsy willbe necessary because of these deficiencies, causing addi-tional expense and anxiety.

As an example of these contentions, it is well knownthat malignant hematolymphoid proliferations and certainmetastatic carcinomas are composed of extremely fragilecells that are exquisitely susceptible to the compressive orshearing effects of some biopsies (Figure 1.1) (2). More-over, it is probable that several cubic millimeters of tissuewill be necessary for the complete pathologic characteriza-tion of such lesions. Hence, a small biopsy specimen wouldbe predictably unsuitable in these circumstances. When indoubt, the clinician should contact the pathologist beforethe procedure is done and inquire about recommendedhandling of the tissue sample and its minimally acceptablesize based on the likely diagnostic possibilities.

Other procedures causing reproducibly detrimentalphysical effects on tissue specimens are represented by

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electrocautery and laser excision. These methods enjoywide clinical usage at present because of their ease of per-formance and the limitation of surrounding tissue damagethat they afford. Nonetheless, lesional cells in the specimenare often rendered unrecognizable because of widespreadthermal coagulation, precluding histologic interpretationaltogether. It should therefore be obvious that cauterizingtechniques must be avoided for diagnostic purposes. Sev-eral adjunctive pathologic studies require the availability ofspecimens that have been handled in a special manner(Table 1.1). Again, these can be obtained prospectivelyfollowing preprocedural consultation with the laboratory.

IDENTIFICATION AND ORIENTATION OF THEBIOPSY SPECIMEN

There is nothing quite so exasperating for the pathologistas to receive a specimen that is unoriented and for which noanatomic location is given on the request form for patho-logic examination. A lack of meaningful clinical historyor a failure to list potential clinical diagnoses often com-pounds such omissions. These problems usually cannot besolved by the pathologist and typically require a laboratoryvisit by, or a telephone conversation with, the responsiblephysician. In many instances, it would be medicolegallydangerous to attempt a morphologic interpretation in theabsence of such information. On occasion, a specimen maybe received that is so poorly labeled that the identity of thepatient is even in question. Such a submission shouldnever be accepted by the laboratory unless the clinician iswilling to provide written documentation verifying its or-igin and accepting exclusive legal responsibility for itsinterpretation.

If a lesion is a suspected malignancy for which a diag-nostic biopsy is also intended to be a complete excision, theclinician should provide some means of identifying thesuperior, inferior, medial, and lateral borders of the tissuesample. This can be accomplished by attaching sutures ofdiffering lengths or types to the specimen, and sendinga corresponding ‘‘map’’ of the tissue to the laboratoryalong with the pathology request form (3). Alternatively,indelible (e.g., tattoo) ink of various colors can be affixed tothe borders of the specimen and identified accordingly(Figure 1.2). As a minimal requirement—for example, invery small excisional biopsies—at least one pole of an el-liptical or circular tissue fragment should be labeled bysuch means.

FIGURE 1.1: Smudging or ‘‘crush’’ artifact results from the mis-

handling of tissue from fragile neoplasms such as lymphoma or

small cell carcinoma (shown here).

TABLE 1.1: Specimen Processing

Pathologic Technique Recommended Fixative Processing Time Comments

Conventional histology NBF or FA* 1 day Tissue should be sectioned at 2–3 mm forgood fixation

Immunohistology NBF or FA** 2–3 days Technique can be applied to frozen or fixedsections

Electron microscopy 2% phosphate-bufferedglutaraldehyde

3–4 days Tissue must be minced into 1- or 2-mm cubes

Immunofluorescence None, if tissue is flash frozen;95% ethanol or acetonefor touch preparations;Michel’s medium for transportation

1–2 days Tissue can be held in Michel’s medium for upto 48 hours. Frozen tissue must be kept at�70�C until use

In situ hybridization NBF or FA for DNA studies; frozentissue preferred for RNA studies

1 week DNA studies can be done on frozen or fixedtissue

NBF = neutral-buffered 10% formalin, FA = NBF-ethanol (50%:50%).

* Tissue for routine histology can be fixed in B5 or Bouin’s solutions to improve nuclear morphology, but these preservatives require special processingand compromise immunohistology.** Certain tissue antigens (e.g., light chain immunoglobulins) are detectable only by frozen section immunohistochemistry.

2 DIAGNOSTIC HISTOCHEMISTRY






The clinician should be discouraged from attempting toprosect the specimen further before it is examined by thepathologist, except in very well-defined settings. Whenthey are improperly performed, transections of small bi-opsy samples often confound subsequent orientation stepsand may mechanically damage the lesion that is intendedfor study. The only acceptable reason for undertaking fur-ther clinical manipulation of the tissue sample is that ofpreparing cellular ‘‘touch’’ preparations in examples ofsuspected hematolymphoid disease. The latter can beobtained if the operator bisects a lesion at its bulkiest pointand touches the cut surface of the tissue gently to glassslides in a serial fashion (Figure 1.3) (2).When this is done,special care should be taken subsequently to orient bothhalves of the resulting two-part specimen for the patholo-gist. Moreover, all air-dried or fixed touch preparationsmust be labeled with the patient’s name, his or her dateof birth or medical record number, and the date on whichthe procedure was performed.

PREPARATION OF FROZEN SECTIONS

Intraoperative consultations, generically termed ‘‘frozensections’’ (FSs) by many surgeons, are often requested intreating presumed or proven malignancies (3,4). Proce-dural aspects of the FS method are familiar to all anatomicpathologists, but these will be reviewed briefly in thissection.

The purposes of obtaining FS examination are twofold;it may be used to secure a rapid diagnosis for a lesion withunknown histologic attributes, or the technique may beemployed to confirm that margins of excision are unin-volved by the pathologic process in question. Because ofthe potential distortion ofmorphologic detail that this pro-cedure may induce, the first of the cited applications is notone that should be used frequently. With respect to theanalysis of excisional margins, the operator must be certainto supervise the orientation and labeling of all specimens,

FIGURE 1.2: Gross room inking stations (A) contain indelible inks of several colors, which can be applied to specimens with cotton-

tipped swabs and fixed in place with Bouin’s solution. The ink can then be seen in an FS (B) or permanent section.

FIGURE 1.3: Touch preparations can be made from fresh tissue

by serially touching a cut surface from it to adhesive-coated glass

slides (A). The touch imprint sections can then be air dried and

stained with Romanowsky dyes, or briefly fixed in alcohol and

stained with H&E (B).

Chapter 1 l Tissue Procurement, Processing, and Staining Techniques 3






as outlined above. This makes the availability of indelibleink an absolute requirement.

Following such steps, one must be certain that the tissuesample is small enough to assure rapid and uniform freez-ing, and ease of sectioning with the cryomicrotome (cryo-stat). The specimen is usually placed in a small pool ofgelatinous, water-soluble mounting medium (e.g., ‘‘opti-mum cutting temperature’’ medium; CryogelR) that hasbeen applied to a precooled Teflon or metal ‘‘chuck.’’ Aftermaking sure that the tissue is properly oriented on the flatsurface of this implement, it is then totally covered withadditional mounting medium, fashioned into a circularpledget. Immediately thereafter, best results are obtainedif the chuck is immersed in a bath of isopentane suspendedin an outer container of liquid nitrogen. These devices areavailable commercially, and they allow for virtually instan-taneous freezing of the mounting medium with minimal-ization of ice crystal formation. The latter eventuality isundesirable because entrapment of ice in the specimen(caused by slowly decreasing temperature) will cause sig-nificant distortion (Figure 1.4) and may interfere with mi-croscopic interpretation. For this reason, the utilization ofmetal cooling ‘‘plates,’’ which are incorporated into manycryostats by their manufacturers, is not recommended asa means whereby initial freezing is accomplished. How-ever, these plates are acceptable for maintaining the chucksin a frozen state while sections are being cut.

The microtome in any cryostat must be set in sucha manner that uniform sections of reproducible thickness(approximately 5 lm) can be prepared. Regular mainte-nance regarding the sharpness and integrity of microtomyblades is essential to this process. After ‘‘facing’’ the frozenblock with the blade—to obtain a smooth, flat tissuesurface—the operator cuts a ‘‘ribbon’’ of several individualsections that can be kept flat by manipulation witha camel-hair brush or with a Teflon-coated panel. These

are then apposed to acid-cleaned glass slides that have beenkept at ambient temperature, causing the tissue to adhereto them quickly. To eliminate concerns about the subse-quent loosening of this bond, slides that have been pre-coated with albumin, poly-L-lysine, or a chrome-alum gelmay be utilized (5).

Most FS laboratories employ a brief (30–60 second)fixation step immediately after mounted sections are pre-pared, in Copland jars containing absolute acetone or 95%ethanol. The slides may then be stained with hematoxylinand eosin (H&E), a ‘‘polychromatic’’ or metachromaticreagent such as methylene blue, or other reagents. Follow-ing dehydration in graded alcohols and xylene, a syntheticmounting medium is placed over the tissue, and a glasscoverslip is applied. Addition of a few drops of xylene tothe mounting medium will slightly lessen its viscosity andhelp to prevent the entrapment of air bubbles under thecoverslip.

Alternatively, one may wish to keep some unstained FSsfor future studies. This aim is best served by removingslides from the acetone or alcohol fixative and placing thempromptly in a freezer at�20 or�70�C.These can be kept insuch devices indefinitely for further analysis at a later date.

Specific problems connected with poor microtomytechnique will be considered subsequently in this discus-sion. However, the most common difficulty that is seen inthe FS area can be ascribed to improper calibration of thecutting interval between successive sections. Overly thicksections may result in consumption of the tissue beforea suitable slide is obtained for microscopic examination;in contrast, it is extremely hard to obtain very thin sectionswithout causing them to fold on themselves or shred.Thus, it is essential for the cryostat to be checked fre-quently to make certain that it is set up properly froma technical viewpoint. Also, there is no substitute for prac-tice and experience on the part of the operator, in regard topreparation of optimal FS slides. The labeling of speci-mens used for FS examination should be no different thanthat used for other samples. The remnant tissue should beplaced in a plastic cassette that is suitably inscribed with theaccession number of the case (preferably using a Cas-MarkR-type labeler) and kept together with correspondingpaperwork for transmittal to the histology laboratory. Un-der no circumstances should unlabeled frozen tissue beallowed to accrue in the FS laboratory, lest disastrouserrors in identification occur.

FIXATION OF SPECIMENS

Questions that are often asked of the pathologist concernthe choice of one fixative solution over another for thepreservation of various cutaneous specimens. There is no‘‘universal’’ fixative in pathology because tissue samplesmay be used for an ever-growing number of investigativeanalyses, many of which demand that special processing

FIGURE 1.4: If ice crystals are allowed to form in tissue during

the freezing process in a cryostat, linear defects will appear in the

final FS.







measures be applied in order to procure optimal results.Selected immunohistologic studies, electron microscopy,and genotypic assessment represent three advancedmodal-ities of pathologic evaluation that are associated with spe-cific fixation requirements. Laboratory specialists arecontinuing to develop procedural modifications to lessenthe need for such provisions, but they still do exist.

In the most optimistic of scenarios, it would be best tosubmit all biopsies in their fresh state in physiologic salinesolution, and for the pathologist to subdivide these speci-mens into several parts for future diagnostic eventualities.Nevertheless, this is often not practical for two main rea-sons. First, outpatient specimens are commonly submittedover long distances from the pathology laboratory, increas-ing the likelihood that unfixed tissue will undergo autolysisbefore it is received. Second, many biopsies are limited insize, making judiciousness in the selection of special studiesan important point. The latter issue again emphasizes thewisdom of preprocedural consultation with the patholo-gist, if unconventional evaluations are desired.

Fixatives Used for ‘‘Routine’’ Histopathologic Examination

In the great majority of cases, the clinician requesting his-tologic examination of a biopsy is interested in a ‘‘tradi-tional’’ interpretation based on microscopic findings asseen with the H&E stains. With this stipulation in mind,most laboratories have advocated the use of formalin as thefixative of choice. Nonetheless, the following sections willbriefly review the chemical characteristics of preservativesolutions in a broader sense, so that exceptions to theabove-cited situation may be addressed.

General Considerations

The preservative effects of certain chemicals have beenrecognized for thousands of years, dating back to the an-cient Egyptians. On an empiric basis, therefore, variousfixatives have been employed to preclude bacterially medi-ated putrefaction of human tissues since the inception ofpathology as a discipline.

In the past century, detailed studies of these agents haveelucidated the probable mechanisms responsible for thesebeneficial effects (6–10). In addition to antibacterial ef-fects, fixatives also enhance the differences in refractiveindices between dissimilar tissue constituents, allowingfor greater resolution upon light microscopy. Moreover,they augment the affinity that chemical dyes have for par-ticular cellular elements. It is now known that chemicalfixatives may be divided into two broad categories—coagulating and noncoagulating—with respect to theireffects on proteins, which form the framework of virtuallyall cells. Further subdivision into aqueous and nonaqueousagents, as well as additive or nonadditive preservatives, isalso possible (11).

Noncoagulative fixatives are the most widely used, andthese include formaldehyde (called formalinwhen preparedin aqueous dilution and paraformaldehyde when employedin polymeric form), glutaraldehyde, acetic acid, potassiumdichromate, and osmium tetroxide. In contrast, acetone,alcohols, chromium trioxide, mercuric chloride, and picricacid exemplify the coagulative preservatives. In the processof denaturation and coagulation, a network of alteredprotein is formed in tissue; in contrast, noncoagulativeagents act to produce a stable intracellular ‘‘gel.’’ Acetoneand alcohol are the major nonaqueous reagents, with mostothers being soluble in water. Additive fixatives reactwith tissues by combining with them chemically, whereasnonadditive reagents (primarily alcohols and acetone) do not.

Various combinations of these chemicals [e.g., formalin-alcohol or Carnoy’s solution (a mixture of ethanol, chloro-form, and acetic acid)] are sometimes utilized as fixativesthat are intended to augment the stainability of predefinedtissue components. Moreover, metal salts—such as thosecontaining zinc and mercury—may be added to aqueoussolutions, as in zinc-formalin or ‘‘B5’’ fixative (a mixture ofmercuric chloride, sodium acetate, and formalin). The ap-parent effect of the latter agents is to stabilize complexesthat are formed by nucleic acid and protein, yielding im-proved preservation of nuclear detail. By convention, manyfixatives are named for the laboratory investigators whodevised them. Thus, one may encounter such designationsas Bouin’s, Hollande’s, Zenker’s, Helly’s, Zamboni’s,Orth’s, and Carnoy’s solutions. Most of these are mixturesof chemicals in different classes or showing differing effectson proteins, with or without metal salts. Selected reagentsin this list will be alluded to later in this discussion.

One important concept to be borne in mind is that allfixatives induce chemical artifacts in tissue sections. Thiseffect has two potential ramifications for pathologists.First, we all become inured to the artifacts that we areaccustomed to seeing with routine use of a particular pre-servative solution; indeed, we may even rely on suchchanges as diagnostic features. Changing the fixative oneuses will also alter the tissue artifacts, often leading to in-terpretative confusion with any given staining method.Second, one artifact produced by a preservative may bedesirable, whereas others are detrimental. For example,B5 solution yields excellent nuclear detail on H&E stains,but it virtually destroys the integrity of some cellular pro-teins that may be the targets of immunohistochemicalstudies (12). Lastly, the optimal period of fixation variesgreatly from one solution to another; tissue placed in for-malin may be allowed to remain in it for days with nocompromise of morphologic features, whereas specimensin B5, Zenker’s, and Bouin’s fixativesmust be transferred toother chemical solutions after predefined periods of timeto avoid a serious loss of cellular definition (7,8). Thus, theultimate choice of a preservative solution is not one to bemade indiscriminately.







Specific Fixatives

Formalin: Formalin represents a 37–40% aqueous solu-tion of formaldehyde, the latter of which is marketed com-mercially in the United States. Because the former reagentis characteristically used at a 10% dilution, the final form-aldehyde concentration is 3.7–4%. Various other chemi-cals have been added to formalin to alter its stability andpreservative capabilities, including calcium chloride, cal-cium carbonate, ammonium bromide, sodium chloride,sodium phosphate, sodium hydroxide, and absolute ethylalcohol. Among these mixtures, that consisting of forma-lin, distilled water, and monobasic/dibasic sodium phos-phate is the most widely employed and is known as ‘‘10%neutral-buffered formalin’’ (NBF). Paraformaldehyde isa polymerized form of formaldehyde admixed with meth-anol; it is generally employed as a fixative for specializedimmunohistologic procedures, particularly when com-bined with periodate and lysine (‘‘PLP’’ solution) (12,13).

Although it is a general-purpose fixative and yields goodmorphologic detail when prepared properly, NBF doeshave some disadvantages in tissue pathology. First, anysolution containing formaldehyde is potentially carcino-genic, and levels of formalin vapor in the ambient air ofthe laboratory must be measured regularly by governmen-tal mandate. The maximum permissible exposure limit forany individual employee is 1 part per million over an8-hour period, as established by the Occupational Safetyand Health Administration (14). Second, poorly preparedNBF, which has been buffered erroneously and has a pHoutside of the physiologic range, may cause unwanted pre-cipitates of ‘‘black acid hematin pigment’’ in tissue sec-tions. The latter has a dark particulate appearance, andmay simulate microorganisms on a histologic slide. Thesetwo possibilities can be distinguished through the use ofpolarization microscopy because hematin pigment is bire-fringent, whereas microbes are not (11). Third, NBF that isallowed exposure to ambient air for prolonged periods oftime (as with large ‘‘batches’’ that are diluted for use in thegross laboratory) will develop high levels of formic acid.The latter is detrimental to protein substructure and mayaccentuate the formation of methylol bonds between poly-peptides. This effect can ‘‘mask’’ proteinaceous epitopesthat correspond to the targets of immunohistologic anti-body reagents (15). Lastly, formalin has a limited capacityfor penetration of bulky pieces of tissue, and specimensfixed in it must be no thicker than 4–5 mm.

Despite these drawbacks, formalin is inexpensive andwidely available, and is therefore ubiquitously employedas the fixative of choice for clinical specimens. Theabove-cited failings of this preservative can be preventedby careful technique in its preparation, adherence to envi-ronmental monitoring requirements, and application ofproper prosection and fixation techniques for the submis-sion of tissue sections. Some laboratories prefer to use

NBF ethanol (mixed in equal volumes) because it affordsa greater degree of tissue penetration than formalin alone.

B5/Zenker’s/Helly’s Solutions: B5, Zenker’s, and Helly’ssolutions were introduced because of their superiority overNBF in the preservation of nuclear detail (7,8). They arefixatives based on the inclusion of mercuric chloride, withor without sodium acetate, potassium dichromate, sodiumsulfate, acetic acid, and formaldehyde as additional constit-uents. Because of the excellent morphologic detail that isachievable with these solutions, many laboratories preferthem for the routine preparation of H&E-stained sections.Nevertheless, there are three distinct disadvantages of B5,Zenker’s, or Helly’s reagent, as compared with NBF. Tis-sue sections must be removed from the former three fix-atives after no more than 8 hours and placed into 70%ethanol; if this is not done, specimens will become ex-tremely brittle and virtually impossible to section (11).Also, the presence of mercuric chloride will cause deposi-tion of pigment in microscopic preparations, which mustbe removed with iodine before final staining proceduresare done. Lastly, mercury-based solutions are powerful co-agulating agents and therefore damage many cytoplasmicproteins. This effect commonly renders tissue sections un-suitable for a variety of immunohistochemical studies (12).

Bouin’s Solution: Bouin’s fixative is again based on form-aldehyde as a major component, together with picric andacetic acids in aqueous solution. Like B5, this reagentaffords excellent preservation of nuclear morphology butsuffers from failings pertaining to brittleness of tissue, pig-ment deposition, and adverse effects on cytoplasmic poly-peptides. In addition, Bouin’s-fixed specimens acquirea yellow color (because of the effects of picric acid) thatmust be removed by postfixation washing in alcohol andlithium carbonate. Bouin’s fixative is preferred for visual-ization of delicate mesenchymal tissues because of its su-perior differentiating abilities in regard to these elements(11). Accordingly, some ‘‘stromal’’ special stains (such asthe Masson trichrome method) are best performed onspecimens preserved in this solution.

Acetone and Alcohols: Acetone and alcohols are rapidlyacting fixatives with good penetration of tissue. They alsoafford better preservation of some cytoplasmic enzymesthan formaldehyde-based solutions do, in paraffin sections.However, two major disadvantages attend the use of theseorganic reagents. They cause striking shrinkage of tissuebecause of their dehydrating effects, thereby altering mor-phologic details appreciably. Also, acetone and methyl orethyl alcohol are relatively expensive, and they require spe-cial storage and inventory procedures because of possibleuse by laboratory workers as inebriants. In current prac-tice, these agents are usually applied only in the fixation of







touch preparations and are not commonly utilized in theprocessing of biopsy specimens. Similar comments applyto Carnoy’s solution, which is constituted by ethyl alcohol,chloroform, and acetic acid.

Decalcifying Solutions: Some biopsy samples may containobvious foci of calcium salts, as suggested by anatomiclocation, clinical findings, or difficulty in performing thebiopsy procedure. In these circumstances, two main meth-ods exist for the removal of such minerals from the speci-men. One employs simple acids (hydrochloric or nitric),which rapidly solubilize calcium deposits. The other tech-nique is based on the ability of certain chelating agents—such as ethylenediaminetetraacetic acid (EDTA)—to ac-complish this task. The second of these methods is muchgentler and does not cause the loss of microscopic detailthat acid decalcification may incur. Fixation is allowed toprogress in concert with decalcification with both acidicand EDTA reagents because they are commercially mar-keted as mixed solutions containing formaldehyde.

Glutaraldehyde: Glutaraldehyde is similar in chemicalactivity to formaldehyde; both cause cross-linkage ofproteins in tissue (7). However, glutaraldehyde penetratesspecimens very slowly, making the size of the tissue samplea critical determinant of fixation with this reagent. More-over, 2–4% glutaraldehyde (representing the usual work-ing concentration) has a propensity to cause brittleness ofspecimens that are immersed in it for more than 2–3 hours;transfer to a buffer solution is absolutely necessary afterthis point. For these reasons, among others, glutaralde-hyde is not used often for the preservation of biopsy sam-ples that are intended for light microscopy. However, it isthe preferred fixative for electron microscopy, whereinspecimens are very small and limited ‘‘hardening’’ of tissuemay actually be morphologically advantageous.

Other Factors Influencing Fixation

As outlined by Carson (11), there are several other consid-erations in the fixation of tissue besides one’s choice ofpreservative solution. These include temperature, size ofthe sample, the volume ratio of tissue to fixative solution,the duration of fixation, and the pH of the solution.

Recently, the rapid but controlled elevation of temper-ature with microwave ovens has been utilized as an in-dependent means of fixation, by coagulation of tissueproteins (16). Surprisingly, this process appears to havelittle if any adverse effect on staining characteristics, evenwith immunohistologic methods. However, it must be em-phasized that careful control is the key to thermal fixation;overheating may completely destroy the specimen if it isallowed to reach an extreme level (e.g., over 65�C). Ina more conventional context, there are really no compel-ling reasons to employ fixative solutions at one tempera-ture versus another.

Specimen size is, in contrast, a potentially crucial factoraffecting quality of fixation, and this determinant goeshand in hand with the volumetric relationship betweena tissue sample and the solution in which it is immersed.Large, extremely thick specimens will be inadequately pen-etrated by most fixatives, allowing autolysis to proceedunchecked in their central areas. This problem results ineventual loss of the unfixed foci during microtomy, yield-ing microscopic sections that resemble doughnuts (Figure1.5). Because penetration is facilitated by minor thermal ormechanical currents in the fixative solution, large speci-mens that are covered with an inadequate volume of pre-servative will predictably be underfixed. An experiencedhistotechnologist typically detects this problem uponattempting microtomy of the tissue and will ‘‘run the spec-imen back’’ for more prolonged fixation and reprocessing.However, this consumes additional time and should beunnecessary.

As noted at several points in the foregoing discussion,there is a maximum recommended period of fixation withseveral preservatives, over which unwanted changes repro-ducibly occur in tissue biochemistry. Overfixed specimensare difficult to cut and often demonstrate alterations inmorphologic definition or antigenic integrity. In contrast,underfixation allows bacterial putrefaction to proceed,similarly damaging the tissue sample. Specimens that areimmersed in the most commonly used preservative—NBF—should ideally be processed further within 8–12hours.

The pH of fixatives is not critical for light microscopy,except that certain unwanted pigmentary deposits maybe seen with unduly acidic preservatives. Nonetheless, hy-peracidity is extremely detrimental to cellular ultrastruc-ture and also to the maintenance of tissue antigenicity

FIGURE 1.5: Inadequate fixation of tissues—especially fatty

ones—will often result in loss of the central areas of tissue blocks

in final microscopic sections because they ‘‘fall out’’ during

processing.







(11,12). For these reasons, it would be wise to control pHwithin the physiologic range during fixation, in the eventthat electron microscopy or immunohistology is necessarydiagnostically.

Tissue Processing and Preparation of

Microscopic Sections

Because most commonly employed fixatives are aqueous innature, the next step in tissue processing is usually that ofdehydration and ‘‘clearing’’ (removal of all water from thespecimen). Graded solutions of ethanol are used for thispurpose, and these must be changed frequently to maintaintheir desiccating properties. A variety of clearing agents areavailable, but the most common are xylene and limonenederivatives. In likeness to the alcohols, such reagents maybe contaminated by water with repeated use and should bemonitored closely for this problem.

Xylene is inexpensive and does not leave a residue onglassware or other instrument parts in the histology labo-ratory. In light of these virtues, it is the most popular clear-ing agent. However, xylene fumes are potentially toxic totechnologists, making careful storage, controlled disposal,and environmental monitoring mandatory. In addition, wehave found that xylene may damage the protein substruc-ture of certain fragile tissue antigens (12). Limonene-typeclearing agents are derived from plants and are thereforebiodegradable. They have a strong odor—like that of lem-ons or oranges—which is alternatively perceived as pleas-ant or noxious by various people. Other disadvantages oflimonenes are that they leave a residue on mechanical tis-sue processors and may sometimes interfere with the ad-herence of tissue sections to glass slides. Themicrotomy ofspecimens cleared in limonenes has been said to be easierthan that encountered with xylene (11).

In the relatively early days of histotechnology, all de-hydration and clearing steps were done by hand. Overthe past 45 years, however, a variety of automatic tissueprocessors have been engineered and marketed. Theseare used widely at present and may be divided into twomain groups—‘‘open’’ and ‘‘closed.’’ Open processorsmechanically transfer baskets containing tissue cassettesfrom one ‘‘station’’ (chemical bath) to another, on a com-puter-driven schedule. The latter may be altered by theoperator to change the time of dehydration, clearing, orother steps. Closed instruments vary the solutions towhich each specimen basket is exposed by pumping chem-icals in and out of fixed chambers, again according to a pro-grammed schedule. In other words, open processors movespecimens, whereas closed processors move chemicalsolutions.

Each of these two types of instruments has advantagesand disadvantages. Open processors show a low incidenceof reagent contamination from one station to another, butthey are subject to the mechanical ‘‘hang-up’’ of specimenbaskets in transit. Closed processors do not suffer from the

latter drawback, but they are subject to chemical carryoverfrom one reagent pumping step to another. This poten-tially compromises the dehydration-clearing sequence. Onbalance, individual experience on the part of technologistsand pathologists ultimately determines which type of pro-cessor will be chosen.

EMBEDDING AND SECTIONING OF

BIOPSY SPECIMENS

The final stations in any tissue processor infiltrate all speci-mens with paraffin or another wax-based embedding me-dium. Thereafter, the technologist removes each biopsy(one at a time) from its metal or plastic cassette and pro-ceeds to embed it in a rectangle of additional liquid wax,with attention to the proper orientation of the tissue sam-ple. The pathologist may direct this process by notching orinking one or several surfaces of the specimen (Figure 1.6)and providing a ‘‘map’’ in accompanying paperwork thatindicates whether these should be placed facedown, faceup,or in parallel with the lateral aspects of the cassette. Suchprovisions are usually necessary only with large pieces oftissue. For example, technologists accustomed to handlingskin biopsies will, as a matter of routine, orient the epider-mis perpendicularly to the bottom of the cassette mold andfacing one of its long sides. If several pieces of tissue areincluded in the same block, these are best arrangeddiagonally.

The embedding step is a potential source of great irri-tation (and medicolegal liability) for the pathologist if it isdone by an inexperienced or careless laboratory worker.With few exceptions, small biopsy specimens that are ori-ented improperly cannot be interpreted microscopically(Figure 1.7), necessitating that the block be remelted and

FIGURE 1.6: This microscopic section of a breast biopsy shows

the intersection of two anatomic planes that were labeled by the

submitting surgeon, and inked with two different colors by the

prosecting pathologist.







re-embedded. This takes time, and in the process of facingthe poorly oriented specimen for preparation of initial sec-tions, valuable tissue may be lost (17).

In order to circumvent embedding difficulties, somepathologists have taken to pre-embedding small biopsiesin agar before they are put in cassettes for fixation. Thisdoes assure proper orientation, but agar will not ‘‘fix’’ inthe same manner that tissue does, nor will it respond sim-ilarly to dehydration, clearing, and infiltration by wax. Allthese factors may cause the tissue to ‘‘pop’’ free of thesurrounding agar after embedding and during tissue sec-tioning, defeating the purpose of the agar impregnationstep altogether. Therefore, we do not advocate this pro-cedure, rather preferring to educate technologists on thedetails of orientation during wax embedding. Even a verysmall biopsy can be appropriately configured in the waxblock, with the use of a magnifying lens or dissectingmicroscope.

Paraffin is still the most widely utilized embeddingmedium, but some laboratories have opted to employ‘‘Carbowax’’ as a substitute. The latter compound is a water-soluble wax, making dehydration and clearing of the tissueunnecessary and allowing for direct infiltration of formalin-fixed tissue with embedding medium in the tissue processor(11). This element of simplicity is attractive, but Carbowaxhas its drawbacks. One concerns the dissolution of the em-bedding medium when microtomized tissue ribbons areplaced in a water bath prior to mounting them on glassslides. This unwanted eventuality makes it difficult for thetechnologist to keep the tissue section flat, resulting in unde-sirable folds in the final stained slide. Second, we have notedirregularities in antigen preservation when Carbowax-embedded tissues are studied immunohistologically. The

temperature of paraffin or Carbowax stations in the tissueprocessor, and at the embedding center, must be monitoredclosely. Overheating the wax will cause unwanted thermalartifacts in the tissue and compromise its cellular detail.Excessively cool wax fails to infiltrate the specimensadequately.

Another class of embedding compounds that is pres-ently in vogue in some centers is represented by polymericplastic resins such as glycol methacrylate or epoxy. Disad-vantages of these compounds include the necessity of cut-ting corresponding tissue sections with a glass or diamondknife microtome, and the requirement for a transitionalfluid, such as propylene oxide, to embed the tissue afterdehydration and clearing (11). Moreover, plastic sectionsare difficult to stain with the same intensity as that seen inparaffin-embedded preparations. The main advantage ofplastic media is that extremely thin, flat sections may beprepared by experienced microtomists, providing exquisitecellular detail. In addition, some enzyme-histochemicalstaining methods that otherwise require the use of FSsare possible with specimens embedded in epoxy or glycolmethacrylate.

Histomicrotomy is a seemingly straightforward pro-cess, representing the cutting of serial paraffin-embeddedsections with a tissue microtome. Nevertheless, this tech-nique has many hidden traps that relate to the propermaintenance, calibration, and orientation of cuttingblades; preparation of paraffin blocks; and dexterity ofthe technologist. Microtome blades that are dull loose ornicked will produce ‘‘chatter’’ or ‘‘venetian blind’’ artifactsin tissue sections (Figure 1.8). In addition, the ‘‘clearanceangle’’ (between the tissue block and the microtome knife)is crucial to good technique. It should be approximately3–8�. If the angle is too narrow, alternately thick and thinsections are cut, or they are folded on themselves (18–20).An excessive clearance angle causes chattered or otherwise

FIGURE 1.7: Malorientation of biopsy ‘‘tips’’ or bisected biopsy

specimens at the embedding station, as shown here, will compro-

mise the pathologists’ ability to evaluate the true specimen mar-

gins for tumor involvement. Reprocessing may or may not solve

this problem, and every effort should be made to avoid it in the

first place.

FIGURE 1.8: ‘‘Chatter’’ artifact in tissue sections is the result of

loose microtome blades or poor microtomy technique.







hideous sections and may preclude the ability of the tech-nologist to obtain a tissue ribbon. Even worse are theeffects of loose microtome blades or tissue blocks in themicrotome chuck. These deficiencies may shatter the par-affin block entirely or deeply groove the tissue specimen. Ablock that is mounted crookedly in the microtome chuckwill produce irregular ribbons or cause individual sectionsin the ribbon to break free from one another.

Regardless of whether one uses paraffin or Carbowax asan embedding medium, there is still a need to refrigeratetissue blocks before microtomy is attempted. This stephardens the wax slightly and allows for crisp sections tobe cut. Warm blocks will yield wrinkled ribbons or causesuccessive sections to anneal to one another. In addition,failure to moisten the surface of blocked tissue suitablybefore cutting it yields an excessive number of knife marksor fragmented sections. The technologist can simply ruba wet finger over the block several times prior to micro-tomy, if the specimen is small. If it is large, and particularlyif the tissue is heavily cornified, a wet piece of cloth orcotton soaked in 5% ammonium hydroxide may be appliedfor 2 or 3 minutes to rehydrate the tissue face (18).

Another problem that is sometime seen at this step is thetendency for ribbons to ‘‘fly’’ onto the knife blade. This isthe result of static electricity between the wax or tissue andthe metal blade, and also may be avoided by slightly moist-ening the knife and the block surface before each ribbon isprepared.

MOUNTING OF TISSUE SECTIONS

The wax ribbon of serial tissue sections can be removedfrom the microtome knife as it is cut, by using a woodentongue depressor blade. In this process, the operator exertsslight traction on the end of the ribbon, stretching it grad-ually over the wooden blade, and subsequently depositingit on the surface of a warmwater bath at the cutting station.The temperature of such flotation devices should be kept at5–10�C below the melting point of the embedding wax. Ifit is too hot, desiccated-looking sections will result; in con-trast, cool flotation baths produce excessive wrinkling ofthe tissue.

To facilitate the process of obtaining a smooth,unwrinkled, paraffinized ribbon of tissue, it can bestretched by slight traction on its ends while floating inthe warm water bath. Also, we have found that addinga few milliliters of ethyl alcohol to the water is beneficialin this regard. The ribbon must not be left in the bath formore than 1 or 2 minutes, or spurious overhydration of thetissue will be produced. This effect simulates the appear-ance of edema fluid microscopically (17). Because tissuesections do not adhere well to untreated glass slides,a bonding agent also must be a component of the waterbath. Elmer’s-GlueR, albumin, and poly-L-lysine are allsuitable additives of this type.

One of the most dangerous of all mistakes in the histol-ogy laboratory can take place when mounting sectionsfrom flotation baths. Friable tissue may ‘‘shed’’ small frag-ments that float free on the surface of the water, and thesemay be inadvertently picked up whenmounting slides fromsubsequently processed unrelated cases. Derisively knownas ‘‘floaters,’’ these rogue pieces of tissue commonly causeagonizing interpretative problems for the pathologist (Fig-ure 1.9). For example, it is not difficult to envision a smallpiece of a prostatic carcinoma that may find its way ontoslides of another prostate biopsy, a ribbon of which ismounted subsequently in the same water bath. Technolo-gists must be impressed with the tremendous medicolegalliabilities that such a mistake incurs, and they must rou-tinely skim, or otherwise clear, the surface of the water bathbetween cases. An alternative source of floater-type arti-facts is the ‘‘tongue blade metastasis,’’ wherein tissueadheres to a wooden applicator stick that is used to floatsuccessively prepared ribbons from two different cases(11). Needless to say, this practice is highly inadvisable.

With respect to optimizing the cost of slide preparation,we recommend that as many individual sections as possibleshould bemounted on one slide, from the same ribbon. It isnot difficult for adept technologists to include three to fivecuts of a specimen on each slide, arranged in a serial fash-ion. Also, in light of the limited size of many skin, bron-choscopic, and gut biopsies, it is advisable to save anyunmounted paraffin ribbons (with appropriate identifica-tion) from such cases for 1 week after they are accessioned.Remounts can be prepared from these directly, without theneed for further microtomy of the tissue block.

Finally, the identification of tissue sections must bescrupulously maintained throughout the remainder oftheir sojourn in the histology laboratory. Such a necessityis assured by having the technologist scratch the case and

FIGURE 1.9: ‘‘Floaters’’ (lower left) in microscopic sections are

unwanted pieces of tissue from other cases. They may cause seri-

ous diagnostic mistakes and again should be avoided at all costs.







CHAPTER 1 Tissue Procurement, Processing, and Staining...

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