Before the Autopsy

The ability to conduct an autopsy safely and competently must be established. Guidelines for doing so have been published by a variety of national oversight organizations such as the College of American Pathologists and the Royal College of Pathologists (United Kingdom). The autopsy area must have adequate space and lighting, adequate temperature control and ventilation, adequate storage provisions (including pre-autopsy refrigeration and post-autopsy retention of tissues), appropriately sized instruments for dissection (which must be clean and sharp), a calibrated scale for reliable weighing of organs, photographic equipment, rigorous safety policies and procedures, the ability to clean and decontaminate the autopsy area, and a procedure for disposal of tissues. For medicolegal autopsies, the facility must be able to ensure a chain of custody of all specimens.

Details of the complete autopsy are beyond the focus of this book. Several other textbooks describe broader approaches to the pediatric autopsy (1–4).

Having determined that an autopsy is needed and has been properly consented or approved, the pathologist should determine in advance what ancillary tests are necessary. In some circumstances, this is simply a matter of ensuring that appropriate specimen containers are available. Additional time and organization will be required to arrange for postmortem imaging (e.g., X-rays, computed tomography scan, or magnetic resonance imaging; see Chapter 2).

Initial Steps of the Autopsy

Important documents can be photographed or scanned and then incorporated into the digital record. Digital photography should be used throughout the procedure and at each step of the dissection. Photographs of the covered and uncovered body, as well as all forms of identification, are needed. Detailed photographs of all external evidence of medical intervention and trauma are particularly important for medicolegal cases. Incorporate a metric ruler into images so that measurements of abnormalities can be verified. If the color of a lesion is important, at least include a ruler with a neutral white background (so that white balancing can be done on the digital images) or ideally a validated color chart.

Routine external body measurements (body weight, crown-rump length, crown-heel length, foot length, occipital-frontal head circumference) must be obtained. A detailed external examination should include evaluation for post-mortem maceration or autolysis, evidence of trauma and medical intervention, muscle bulk, limb position, pallor, dysmorphic features, and head shape.

Opening the Skull

The nervous system can be examined before or after internal examination of the abdomen, chest, and neck. The decision should be made based upon the emphasis of the autopsy. For example, if clinical data indicate that the nervous system is likely the major site of pathology, it should be examined first to avoid the introduction of artifacts. If a cerebrospinal fluid (CSF) sample is required (e.g., for microbial culture), this should be obtained aseptically with a sterile needle and syringe from the lumbar spinal canal. CSF can also be obtained from the cisterna magna by hyperflexing the neck and inserting a needle through the foramen magnum in the midline, or by insertion of a needle through the anterior fontanelle along the medial surface of a cerebral hemisphere.

The scalp incision should extend in the coronal plane behind the ears and curve slightly forward, approaching the vertex of the skull, to allow reflection of the scalp forward and backward. Extending the incision toward the occipital protuberance and then inferiorly along the spine in a question mark shape (Figure 9.1) allows the occipital bone to be removed for a detailed examination of the posterior fossa. This is advantageous in small fetuses and in cases with suspected malformations of the cerebellum and/or brainstem.

Figure 9.1 Diagram showing path of the scalp incision along the posterior aspect of the head to allow brain removal. Bicoronal incision from A to B posterior to the ears and extending over the vertex of the skull allows removal of the brain from the top. Extending the skin incision along C allows removal of the occipital bone and detailed examination of the posterior fossa.

In routine autopsy cases of infants, the bicoronal scalp incision may be used. Use gentle blunt dissection to separate the scalp from the galea aponeurotica. Reflect the scalp flaps anteriorly over the face and posteriorly over the occiput (Figure 9.2). Inspect and measure the calvarial sutures, in particular the anterior fontanelle. In fetuses and neonates with open sutures, incise the fontanelle and with strong curved scissors follow the coronal sutures down to the temporal bones. The dura mater including the superior sagittal sinus are tightly adherent in the midline at this age. If it is necessary to inspect the sinus (e.g., in a case of suspected obstetrical trauma) (5–7), carefully incise the soft tissues of the sagittal suture as far lateral to the midline as possible (Figure 9.3). Cutting the coronal and lambdoidal sutures allows the parietal bones to be reflected laterally, away from the frontal and occipital bones (Figure 9.4). In older infants (e.g., >6–9 months) with a thicker skull and partially fused sutures, this procedure might not be possible. In these cases, a circumferential saw cut through the calvarium in a horizontal plane above the eyebrows and ears to the occiput will be necessary (Figure 9.5), as is done in adult autopsies (2). When the cranial sutures are almost closed, the dura mater is more easily separated from the inner table of the skull.

Figure 9.2 Photograph showing scalp reflected anteriorly and posteriorly to reveal the coronal (Cor) and lambdoidal (Lam) sutures.

Figure 9.3 Photographs showing stepwise opening of the skull of a 6-week infant with preservation of the superior sagittal sinus. First, a scalpel is used to incise the dura mater at the anterior fontanelle (top panel, arrows). Next, the dura and cranium are cut with scissors on both sides, parallel to the sinus (middle panel; arrows show dotted lines). Finally, the frontal and parietal bones are reflected laterally, leaving the sinus and falx cerebri (lower panel, arrows) attached to the brain.

Figure 9.4 Diagram showing vertex view of fetal or neonatal skull and brain demonstrating “flower petal” opening of skull. Incisions are made along the entire lengths of the coronal and sagittal sutures using a scalpel or scissor. Using strong curved scissors, the parietal bones (Par) are split in the coronal plane down to the temporal bone. Manual fracturing of the parietal-temporal sutures allows lateral reflection of the parietal bones away from the frontal (Fr) and occipital bones (Oc). With this method, the bones of the calvarium remain attached to the skull base. In some cases, it is helpful to (a) split the metopic suture in the midline of the frontal bone, which improves visualization of the orbital surfaces, and (b) to trim the occipital bone, which allows easier opening of the tentorium cerebelli. Positioning the occiput downward, gravity and careful blunt dissection will separate the brain from the skull base.

Figure 9.5 Photographs showing the exposed skull of a 1-year-old infant (upper panel; arrow = coronal suture). An angulated circumferential saw cut (dashed line) allows removal of the calvarium to expose the dura mater (lower panel; arrow = middle meningeal artery), which can then be incised and reflected to expose the brain.

In fetuses, and in infant cases wherein the posterior fossa contents are of particular interest, the posterior approach with occipital bone removal should be used (Figure 9.6) (8).

Figure 9.6 Photographs showing exposure of brain of 26-week fetus after removal of the occipital bone (upper panel). The falx cerebri (F) and tentorium cerebelli (T) are visible. The second step involves reflection of the parietal bones to expose the cerebral hemispheres (lower panel).

Brain Removal

When opening the skull, damage to the meninges should be avoided; this is not always possible. The brain should be examined and photographed in situ. Intracranial blood collections should be photographed in situ and, if possible, the volume measured or at least estimated through measurements in three dimensions. Because fetal and infant brains are very soft, they can be easily damaged during removal from the skull. Therefore, immersion of the head (or entire fetus) in cool water is advised (9) (Figure 9.7). This minimizes abrupt movements of brain and offers some buoyancy. The brain can be slowly poured out of the skull, gradually exposing and incising cranial nerves, arteries, and the tentorium (10). The spinal cord can be severed last from the anterior approach if the calvarium has been removed. If the posterior fossa approach has been used, the spinal cord and tentorium are incised first, followed by tipping the brain out of the posterior fossa. For some malformations, it may be desirable to remove the brain and spinal cord together (Figure 9.8). The brain must be weighed after removal of the dura mater.

Figure 9.7 Photograph showing removal of the brain of a 25-week fetus under water. The brain is gently separated from the base of the skull (arrow).

Figure 9.8 Photographs showing removal of intact brain and spinal cord from a 22-week fetus. After exposure of the posterior fossa and spinal cord (left panel), the parietal bones are reflected. Then the brain is removed from the skull beginning at the frontal lobes. The cranial and spinal nerves are carefully dissected, taking care not to bend the spinal cord. After fixation in formalin, the specimen (right panel) can be examined in detail.

Increasingly with public concerns about organ retention, compromises may be necessary. Selective sampling of brain parts identified as being abnormal on pre- or post-mortem imaging can be done (11). If a metabolic disorder is under consideration, muscle, heart, brain, and liver samples should be frozen at –70˚C and processed for electron microscopy (12).

After removal of the brain, the inner surface of the calvarium and skull base should be examined. In trauma cases, the dura mater must be stripped away to allow inspection for fractures or abnormal suture mobility. Removal of orbital plates from the anterior fossae allows inspection of the orbit contents and eyes. If infection is suspected, the temporal bones should be opened to allow inspection and microbial sampling of the inner ear. The pituitary should be removed from the sella turcica and placed in formalin.

Fixation of the Brain

The brain is best examined after fixation in formalin. Oehmichen and coauthors warned, “The brain must not be examined fresh, or roughly 50% of lesions will be missed, compromising further examination, referral and ultimately the veracity of evidence presented in court” (13). If there is a prohibition against this (e.g., parental request, religious decree), the brain can be sliced fresh and sampled widely for histology.

Chemical fixation is most commonly achieved by immersion of the brain in a 10 or 20% solution of neutral buffered formalin. Some authors advocate for the addition of 5% acetic acid to formalin of use of Bouin’s fixative (picric acid, acetic acid, and formaldehyde in an aqueous solution) (14). Both make the brain firmer than 10% neutral buffered formalin alone. However, many laboratories eschew picric acid because it is volatile and an explosion hazard. Furthermore, tissues fixed in Bouin’s solution develop a bright yellow color.

The volume of fixative should be at least 10 times the volume of the brain; otherwise, the formaldehyde will be exhausted. All but the smallest fetal brains should be laid in a bedding of cotton/wool batting to minimize distortion. Formalin typically penetrates brain tissue at a linear rate of approximately 0.5 to 1 cm per day (15, 16). Therefore, the target duration for complete fixation will depend on the size of the brain. In cases where early return of the brain is requested, 1–2 days fixation will stiffen the outer tissue sufficiently to improve the quality of examination over no fixation (Figure 9.9) (17).

Figure 9.9 Photographs showing the normal brain of a 4-month infant after 2 days fixation. The outer surface of the brain appears well fixed (upper panel). Coronal slices through the brain (lower panel) show that the formalin has penetrated approximately 1 cm, with deep tissue remaining unfixed, pink, and soft.

Several methods have been described to accelerate the fixation process. These include intraarterial perfusion (18, 19), heating for 6 hours using microwave irradiation (20), injection of formalin into the ventricles prior to autopsy (21), and infusion of fixatives other than formalin into the subarachnoid space under negative pressure (22, 23). None are entirely reliable or practical.

Sampling of the brain for histology is described in Chapter 13. Tissue retention after sampling should be done in accordance with local laws and guidelines. In forensic cases where the brain findings have a bearing on the cause of death, retention until the criminal matter is settled might be reasonable.