Microscopic Examination

Tissue Fixation

Unlike tissues obtained from surgery, autopsy material has generally undergone some postmortem autolysis, the degree influenced by the time interval since death, body size, conditions in which the body has been stored, and to a certain extent the underlying disease processes. The most important variable is how soon the internal organs cool to refrigerator temperature. In view of this, careful handling and fixation of tissues obtained at autopsy are important to avoid further tissue degradation. First, one should avoid excessive handling or forceful rinsing of tissues, particularly those with delicate mucosal membranes. It should be noted, however, that gentle rinsing of tissues with water probably does little damage to histologic preparations of autopsy specimens. Second, the tissues should not be allowed to dry out. Third, the tissues should be fixed in an adequate amount of fixative. Save small (i.e., 3 × 3 × 0.5 cm) pieces of representative tissues and specific lesions for microscopic examination in an adequate amount of fixative, typically at least 10 volumes of fixative to 1 volume of tissue. For gross pathology demonstrations, whole or large portions of organs may be fixed and then stored in less than the optimal amount of fixative. Finally, large collections of blood should be gently removed from the external surfaces of tissue (e.g., circle of Willis in case of vessel rupture) before fixation. Not only does this improve visualization of the anatomy, but it also facilitates fixation. Bloody fixatives fix poorly. If the fixative becomes heavily contaminated with blood from congested postmortem tissues, it should be replaced with fresh solution.

The aims of chemical fixation include arresting autolysis so that gross organs retain their shape and microanatomy is preserved and preventing postmortem bacterial overgrowth in preparation for tissue processing and subsequent staining reactions. Typically, 10% neutral buffered formalin (about 4% formaldehyde) has been the most commonly used fixative in autopsy practice. The advantages of formaldehyde-based fixatives include relatively rapid tissue penetration and thus good preservation of cell organelles, limited tissue shrinkage, and minimal tissue hardening. Formalin-fixed tissues are also still suitable for many immunohistochemical and molecular studies. However, biosafety concerns, environmental protection, and cost of disposal have heightened interest in the development of formalin-free fixatives. Most of these are alcohol-based. Compared with formaldehyde, alcohol has the advantage of lower toxicity but the disadvantages of causing increased tissue shrinkage and brittleness. A number of commercially available alcohol-based fixatives contain additives to counter these disadvantages. Environmental and biosafety concerns limit the usefulness of aqueous fixatives based on mercuric chloride (Zenker, Helly), picric acid (Bouin), or chloroform (Carnoy).

Microwave energy speeds fixation by aldehyde or alcohol fixatives and has been used successfully for diagnostic work. Optimal microwave fixation occurs at temperatures between 45°C and 55°C, but the ideal conditions for the tissue of interest must be worked out for each model. Underheating renders soft, poorly fixed tissue, making sectioning difficult. Overheating produces tissue vacuoles, pyknotic nuclei, and overstained cytoplasm, making microscopic interpretation difficult. To cool the tissue after microwaving, the heated fixative should be replaced with fixative at room temperature as soon as possible.

It has generally been recommended that whole brains remain in fixative for 2 weeks before cutting. This practice lengthens the time needed to complete the examination and final report, however. To shorten the time needed to fix brains before optimal sectioning for gross and microscopic examination, Boon and colleagues used microwave energy in combination with formalin- or alcohol-fixation. Garzón-de la Mora and colleagues have explored electrochemical methods for rapidly fixing human brains. Others have fixed the brain by vascular perfusion either by positive pressure through a syringe or electric pump or by an apparatus employing gravity. Compared with fixation by immersion, perfusion fixation shortens the interval required for fixation of the deep-seated regions of the brain. This not only allows the brain to be cut sooner but also improves immunohistochemical staining. However, in a few brains fixed by perfusion, we have noted an artifact—irregular white matter pallor seen by hematoxylin and eosin stain. Although this appearance simulated white matter disease, there was no attendant gliosis to signify antemortem injury. Finally, vascular diseases such as atherosclerosis or injuries such as inadvertent laceration of the circle of Willis during brain removal can make perfusion difficult or even impossible. In cases of suspected cerebral emboli or thrombi, perfusion fixation of the brain is contraindicated.

If the brain is fixed by simple immersion, it is important to use adequate fixative for this large organ, particularly if the brain is bloody. Some institutions use a large reservoir with circulation achieved by pumps or stir bars. Others will merely exchange the formalin for fresh formalin after several days. When fixing the brain, it is immersed basal surface up with a string slipped under the basilar artery. The string is then run to the lid closure seal to gently suspend the brain just below the surface so that it does not touch the bottom or sides of the container.

Immersion fixation of the fetal or neonatal brain can be facilitated by adding additive-free salt to the fixative. This “floats” the brain, minimizing artifacts produced by compression of the soft unmyelinated brain tissue against the walls of the vessel. Some pathologists fix the fetal or neonatal brain and spinal cord in situ by percutaneously injecting fixative into the lateral ventricles. The advantage of this technique is that it preserves delicate pathologic anatomy in cases of hydrocephalus or porencephalic cysts. Because the injection can be done shortly after death and without any disfigurement, in situ fixation of the central nervous system may also be useful in cases in which autopsy permission has been obtained but postmortem dissection is delayed, particularly in situations in which special diagnostic studies such as electron microscopy or in situ hybridization are required.

Decalcification

Bone and other mineralized tissues contain insoluble calcium salts that make them too hard to cut with an ordinary microtome; they must be softened or decalcified. This is accomplished by chemical removal of the calcium salts in solutions of acid, chelating agents, or ion exchange resins. The decalcifying solutions are generally categorized as: rapid, strong acid (5% to 10% nitric acid or hydrochloric acid); moderate, weak acid (usually 5% to 25% formic acid, sometimes trichloracetic acid or picric acid); or slow, chelating (ethylenediaminetetraacetic acid [EDTA]). The slow, chelating agents are used only for trace levels of calcification. The rapid, strong acids can impair staining but are often needed for large bone specimens, such as large samples from skull base, pelvis, or long bones. The moderate, weak acids are quite useful for thin vertebral body slices or calcified coronary arteries. The decalcification step is performed only after adequate fixation; otherwise, the decalcifying solutions denature the tissue proteins, adversely affecting the histology. To ensure proper fixation and subsequent ease in decalcification, small tissue samples approximately 4 mm thick should be prepared using a bone knife or saw whenever feasible. The fixed tissue should be washed in running tap water to completely remove fixative if subsequent hydrochloric acid decalcifying solution is used, to avoid formation of toxic bis-chloromethyl ether. In contrast, formic acid decalcifying solutions can be combined with formalin. Next, the tissue should be placed in abundant decalcifying solution (as directed by the manufacturer). Changing the solution each day will accelerate decalcification. Suspending the tissue (e.g., by wrapping it in gauze) ensures adequate decalcification of all surfaces of the sample.

Testing the softness of an additional small piece of similar tissue placed in the decalcifying solution is a convenient way to check the progress and yet preserve the integrity of the specimen. Samples that are cut easily with a scalpel will be cut readily on a microtome. Alternatively, one can insert a narrow-caliber needle into several representative regions of the specimen itself. If any grittiness is detected, decalcification is incomplete. The extent of decalcification can also be detected radiographically or chemically. These methods are rarely required, and then only for samples that are difficult to decalcify, such as temporal bones. For chemical assessment of decalcification, aspirate 5 mL of decalcification fluid from the bottom of the container in which a sample has been suspended for at least 6 hours and in only 5 times the volume of the specimen to ensure that any calcium is in high concentration. Then add 5 mL each of 5% ammonium hydroxide and 5% ammonium oxalate and mix well. Formation of a cloudy precipitate indicates incomplete decalcification. If the solution is clear, decalcification is complete. After determining that decalcification is adequate, the tissue is placed in a labeled cassette and rinsed well under running tap water. The rinsing removes the decalcifying solution and allows adequate processing. After rinsing, samples are stored in fixative until final tissue processing.

In cases of metabolic bone disease, alternative methods may be preferable to standard processing. For example, embedding in plastics (methyl methacrylate, glycol methacrylate, or a combination of the two) and preparation of sections with glass knives allow examination of nondecalcified bone. Specialized grinding techniques allow the cutting of large sections.

General Guidelines for Microscopic Sampling of Tissues

In preparation for sampling for histology, trim the fixed tissues carefully. Use a sharp scalpel, and do not crush the tissue when cutting. Cuts should be made with smooth strokes of the blade in one plane, rather than chopping or short sawing motions. Cut tissue to a thickness of approximately 3 mm, or less if the tissue is particularly dense or fatty. Forceps with wide paddles at the end are very helpful when bisecting tissues to the correct thickness. Place the best flat face down in the cassette, because that will be the face cut during microtomy. Do not overload the tissue cassettes because compressed tissues cannot be adequately dehydrated, infiltrated with paraffin, or properly embedded for optimal sectioning. Putting tissues of similar density together in cassettes makes for easier sectioning. If labeling cassettes by hand, pay particular attention to numbers such as 4 and 9 and letters C and G; D, O, and Q; and U and V, which can resemble each other. For more clarity and flexibility of labeling, most institutions are switching to printed cassettes with sequential numbers such as A1, A2, and so on, often with barcodes. Box 8-1 lists standard sections (excluding the central nervous system) that are submitted from pediatric and adult autopsies in our university hospitals; additional sections from areas of pathology are usually warranted. Box 8-2 and Figure 8-1 provide guidelines for microscopic sampling of the brain and spinal cord.

Except as indicated in Boxes 8-1 and 8-2 , there are no set rules for determining where tissue from normal-appearing organs should be selected for microscopy. Whenever possible, however, organ capsules are sampled along with the underlying parenchyma and serosal or adventitial surfaces along with mucosa or endothelium. In other words, full-thickness sections should be taken from hollow viscera, if possible. Microscopic sections of the kidney include cortex, medulla, and calyx. To take samples of bone for decalcification, a hammer and bone knife are preferable to a bone saw, which leaves bone dust as an artifact. If a bone saw is used, be sure to use a scalpel to cut a fresh dust-free face after decalcification before submitting for histology. Careful dissection, visual examination, and knowledge of pathologic processes guide the autopsy pathologist in the selection of sites and extent of sampling for microscopy. For example, sample primary malignancies in such a way that tumor progression and stage can be assessed. For discrete lesions, sample the edge of the lesion along with the adjacent transition into “normal” tissue, because this demonstrates the host response to the injury, often useful in dating the onset of the disease process. A number of manuals of surgical pathology offer guidelines for tissue sampling of individual organs and provide useful suggestions for microscopic sampling of organs.

Specialized Microscopic Examination of the Brain in Dementia Cases

Many autopsies are now performed primarily to assess the cause of dementia or neurogeneration—with some of those restricted to examination of “brain only.” A consensus report from the National Institute of Aging recommends that “Alzheimer disease neuropathologic changes” may be diagnosed based on the presence and extent of brain lesions seen at autopsy, regardless of cognitive state. Given that other diseases may create similar histologic findings and several diseases may be present in some individuals, a thorough analysis requires sampling many areas of the formalin-fixed brain with examination of paraffin sections for (1) assessment of amyloid plaques, (2) staging of neurofibrillary tangles, and (3) scoring of neuritic plaques—along with assessment of Lewy bodies, vascular brain injury, and hippocampal sclerosis. The preferred method for assessing amyloid plaques and neurofibrillary tangles has shifted to immunohistochemistry in many laboratories. Areas of the brain to be sampled include the standard sections outlined in Figure 8-1 , along with additional sections from anterior cingulate, amygdala, middle frontal gyrus, superior and middle temporal gyri, inferior parietal lobe, and occipital cortex, as outlined in the published guidelines. Gross inspection should assess cerebrovascular disease, regional atrophy patterns, and presence of focal gross lesions. If experts in this field are not available at an institution, it is often prudent to send the whole brain—after 2 weeks of fixation—to an expert for consultation.