Selection Of Tissue Blocks For Histologic Examination

BRAIN AND SPINAL CORD When the lesions in the brain are obvious, selection of the appropriate blocks is simple. For orientation and for possible evidence of pathologic involvement, some recognizable structures from the surrounding and presumably normal areas should be included. When gross lesions cannot be found despite the presence of clinical neurologic signs or symptoms, one must be familiar with the topographic distribution of the lesions expected in a given disease or syndrome to be able to select appropriate sections. Familiarity with the patient's clinical history must be accompanied by some basic knowledge of where the lesions are to be expected.

It is difficult to define what constitutes adequate selection of sections in "routine normal cases." No universally accepted standards exist, but whatever choices are made, selections should be consistent topographically. The areas shown in Fig. 6-15 are our minimal requirements; the reasons for this selection are given in the legend. It is best to store the whole brain until the microscopic examination is completed and the clini-copathologic correlation is satisfied.

In most cases, the size of the sections can be limited to be suitable for the standard 1- by 3-inch glass slides. We use tissue capsules of different sizes for automatic processing machines. We try not to "mutilate" the original brain slices and therefore, if photographs are taken of crosssectional surfaces, we select tissue blocks from the same surface of the adjoining slice so that photography can be be repeated. Alternatively, the brain slab can be sliced thinly up to the area of block removal while the knife blade protects the lower half of the slab and vertical cuts are made into the upper slab. Experienced prosectors can prepare complete thin slices and lay them on the cutting board before blocks are removed. It is not a good practice to hold a thick slab in the hand and to try to undercut a centrally located block through one of the vertical cuts, as this will invariably result in an uneven "dig" into the remaining tissue.

In the absence of known spinal cord abnormalities, one section each from the cervical, thoracic, and lumbosacral levels is appropriate. When spinal cord lesions are expected, patholo-gists should attempt to localize the "radicular-segmental" or "vertebral-body" level of the lesion. Keeping in mind that the conus medullaris generally ends at the level of the upper part of the L2 vertebral body, the Ll and L2 dural root exits can be localized. Cephalad from this point, spinal cord roots and vertebral body levels can be counted. For correct localization of the levels, the dural sac and the exit zones must be intact (see "Removal of spinal cord" and "Dissection of the spinal cord").

PERIPHERAL NERVES The cervical and lumbar plexuses can be removed totally and in continuity with the spinal roots and ganglia, as outlined for removal of the spinal cord. As a routine procedure, this is too time-consuming.

A quicker method is to cut the nerves as they emerge from the intervertebral foramina and to sample selected nerves as the clinical signs dictate. Routinely, lengths of the sciatic and femoral nerves or any other portions of the lumbosacral plexuses proximal to their exits from the pelvic and abdominal cavities can easily be removed without creating new incisions. Similarly, sampling of the brachial plexuses and their distal extensions can be achieved from the supraclavicular axillary regions. Care should be exercised to preserve the brachial arteries for embalming.

In cases in which detailed clinical studies were performed on the peripheral nervous system, the affected nerves should be sampled at autopsy. When incisions are made in the extremities for sampling of muscles, as described in the next section, the nerves innervating them can be removed conveniently. In a diffuse neuropathic condition, one may select the sciatic nerve and its distal ramifications for detailed studies. To this end, the body is turned over and an incision is made in the back of the thigh to free the sciatic nerve, which has been severed previously at its pelvic exit. The incision may be extended caudally to allow the removal of the peroneal and tibial nerves in the leg. More conservatively, a 15-cm longitudinal incision in the popliteal region exposes these nerves at their bifurcation. The arteries in the vicinity must not be lacerated, as this would interfere with the embalming procedure. To assist the embalmer, we have also removed the sciatic nerve by incising the anterior surface of the thigh and leg. Sawing away a portion of the pelvic bone (mainly the ischium) helps to free the nerve without pulling it up or down behind the bone. This approach is cumbersome, but a bonus is the easy removal of the femoral nerve and its branches.

One of the most accessible peripheral nerve is the sural nerve, which has been biopsied in many clinical studies. Therefore, its removal at autopsy through a small incision behind the lateral malleolus gives an excellent base for comparison. For removal and fixation techniques, seeref. (27). A useful adjunct to diagnostic studies of the peripheral nervous system is a fiber-teasing method that has become a standard procedure in many research laboratories. After fixation, a portion of nerve is stained with 1% osmium tetroxide and macerated in 60% glycerol, and individual fibers are teased out under a dissecting microscope. This method allows to examine fibers three-dimensionally and to evaluate axonal degeneration and demyelination (27). For best preservation of these nerves, autopsies should done within 6 h after death.

SKELETAL MUSCLE In the absence of specific diseases affecting the neuromuscular system, skeletal muscle is rarely sampled. One or two specimens should be stored in the "routine" autopsy. The ileopsoas muscle is easily accessible and shows the effects of general systemic disease on the skeletal muscles.

In cases of known or suspected neuromuscular diseases, more extensive sampling is required. For primary myopathies, the selection has to be based on clinical findings and the status of the muscles at the time of autopsy. Sections should be taken from muscles that are severely affected, that show early but active involvement, and that are grossly uninvolved. A list of muscles to be sampled in cases of neurogenic muscle atrophy is shown in ref. (28). Table 6-1 lists muscles that are accessible without major procedures and will give an adequate diagnostic sampling.

The specimens should be cleanly excised or neatly trimmed to about 3.0 x 1.0 x 0.5 cm. Placing the samples on a piece of cardboard does not completely prevent shrinkage of the tissue during fixation. A corkboard with two narrow strips of cork fastened to it provides ridges to which multiple muscle samples can be pinned. This eliminates the problem of poor fixation of the underside of the specimens. Parts of wooden applicator stick may be used to support smaller pieces of muscle, which

Table 6-1

Suggested Muscles for Sampling at Autopsy

Muscle

Comment

Extraocular muscles

Tongue

Sternocleidomastoid; diaphragm; pectoralis major

Biceps; triceps

Forearm muscles

Intercostal;

psoas major Quadriceps

Anterior tibialis; gastrocnemius

Obtained through orbital plate intracranially or anteriorly with or without the globe.

Removed with pharynx and larynx; small pieces can be removed through mouth.

No new incision required; pectoralis major is preferred over deltoid because previous intramuscular injection into deltoid may have caused abnormalities.

Removed through incision in axillary aspect of upper arm or by subcutaneous extension of primary incision into arm.

Morticians generally consider skin incision on the forearm undesirable, particularly in females. Incision in ulnar side of palmar aspect of forearm is least objectionable.

No new incision required.

Removed through incision in ventral aspect of thigh.

Removed through incision in lateral aspect of lower leg.

are tied to it with suture material at both ends. We consider 10% neutral formalin solution the most satisfactory all-purpose fixative, particularly if staining of the nervous tissue in the specimen is important. Additional pieces can be fixed in Bouin's solution to improve trichrome stains; fresh-frozen cryostat sections can be prepared for Gomori's trichrome stain and for staining with hematoxylin and eosin, after 2 min fixation in 10% formalin solution on a cover slip (Engel AG, personal communication).

Teasing the removed specimens lengthwise after fixation, rather than cutting with a knife blade, sometimes produces a better longitudinal arrangement of the muscle on histologic slides. As with peripheral nerves, both cross and longitudinal aspects of the muscle should be represented.

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